Antisense modulation of BH3 interacting domain death agonist expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of BH3 Interacting domain Death agonist. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding BH3 Interacting domain Death agonist. Methods of using these compounds for modulation of BH3 Interacting domain Death agonist expression and for treatment of diseases associated with expression of BH3 Interacting domain Death agonist are provided.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/657,346 filed on Sep. 7, 2000.

FIELD OF THE INVENTION

[0002] The present invention provides compositions and methods formodulating the expression of BH3 Interacting domain Death agonist. Inparticular, this invention relates to compounds, particularlyoligonucleotides, specifically hybridizable with nucleic acids encodingBH3 Interacting domain Death agonist. Such compounds have been shown tomodulate the expression of BH3 Interacting domain Death agonist.

BACKGROUND OF THE INVENTION

[0003] Apoptosis, or programmed cell death, is a naturally occurringprocess that has been strongly conserved during evolution to preventuncontrolled cell proliferation. This form of cell suicide plays acrucial role in the development and maintenance of multicellularorganisms by eliminating superfluous or unwanted cells. However, if thisprocess goes awry, excessive apoptosis results in cell loss anddegenerative disorders including neurological disorders such asAlzheimers, Parkinsons, ALS, retinitis pigmentosa and blood celldisorders, while insufficient apoptosis contributes to the developmentof cancer, autoimmune disorders and viral infections (Thompson, Science,1995, 267, 1456-1462).

[0004] The Bcl-2 family of proteins, which includes both positive andnegative regulators of apoptosis, act as checkpoints upstream ofactivated protease cascades orchestrated by caspases and are requiredfor all aspects of cell death (Chao and Korsmeyer, Annu. Rev. Immunol.,1998, 16, 395-419; Kelekar and Thompson, Trends Cell Biol., 1998, 8,324-330). The Bcl-2 proteins share conserved regions of homology knownas Bcl-2 homology domains or BH domains, four of which have beenidentified to date. It is through the interaction, via dimerization withother Bcl-2 members, of one or more of these domains that the familymembers exert their pro- or anti-apoptotic effects (Chao and Korsmeyer,Annu. Rev. Immunol., 1998, 16, 395-419; Kelekar and Thompson, TrendsCell Biol., 1998, 8, 324-330).

[0005] Anti-apoptotic members of the family include Bcl-2, Bcl-x_(S),Bcl-x_(L) and Bcl-w while pro-apoptotic Bcl-2 members include Bax, Bik,Bid, Bim, Hrk and Blk (Kelekar and Thompson, Trends Cell Biol., 1998, 8,324-330). Three of the pro-apoptotic proteins, Bad, Bid, and Bim, showlittle similarity to Bcl-2, containing only one BH3 domain (Kelekar andThompson, Trends Cell Biol., 1998, 8, 324-330). Disclosed in the PCTapplication WO 99/16787 are the polypeptide and polynucleotide sequenceof the BH3 domain found in Bcl-2 family members, specifically BID, andmethods to promote apoptosis in a cell by administering an effectiveamount of the BH3 domain peptide (Korsmeyer, 1999).

[0006] Bid (also known as BID or BH3 Interacting domain Death agonist)is a member of the Bcl-2 family and has been shown to dimerize witheither Bcl-2, a cell death antagonist, or Bax, a cell death agonist, andcan be found in both cytosolic and membrane fractions (Wang et al.,Genes Dev., 1996, 10, 2859-2869).

[0007] Upon cell surface signaling by a death receptor, it is known thatBH3 Interacting domain Death agonist is cleaved by caspase 8 and theC-terminus translocates to the mitochodria and triggers cytochrome crelease (Gross et al., J. Biol. Chem., 1999, 274, 1156-1163). It is nowknown that this process is mediated by the binding of BH3 Interactingdomain Death agonist to Bax, with the concomitant induction of astructural change in Bax (Desagher et al., J. Cell. Biol., 1999, 144,891-901) and is diminished by binding to Bcl-2 (Luo et al., Cell, 1998,94, 481-490).

[0008] Due to the integral role played by BH3 Interacting domain Deathagonist in apoptosis, the pharmacological modulation of BH3 Interactingdomain Death agonist activity and/or expression may therefore be anappropriate point of therapeutic intervention in pathological conditionsinvolving deregulated cell death. Disclosed in the PCT publication, WO00/11162 is a novel form of BH3 Interacting domain Death agonist (p15BID) created by the selective cleavage of the cytosolic BH3 Interactingdomain Death agonist protein. This 15 kD polypeptide, once cleaved,translocates to the mitochondria where it resides as an integralmembrane protein and is required for the release of cytochrome c (Grossand Korsmeyer, 2000). Also disclosed are uses of p15 BID and mutant p15BID polypeptides for the modulation of apoptosis.

[0009] Currently, there are no known therapeutic agents whicheffectively inhibit the synthesis of BH3 Interacting domain Deathagonist and to date, investigative strategies aimed at modulating BH3Interacting domain Death agonist function have involved the use ofantibodies, molecules that block upstream entities such as caspaseinhibitors (Sun et al., J. Biol. Chem., 1999, 274, 5053-5060) and geneknock-outs in mice (Yin et al., Nature, 1999, 400, 886-891).

[0010] Disclosed in U.S. Pat. No. 5,955,593 and the PCT application WO98/09980 are the peptide and nucleic acid sequence of human BH3Interacting domain Death agonist as well as antibodies, vectors and hostcells used to express the BH3 Interacting domain Death agonist proteinand reporter constructs used to detect said expression (Korsmeyer, 1999;Korsmeyer, 1998). Antisense oligonucleotides complementary to BH3Interacting domain Death agonist 15 to 30 nucleotides are also generallydisclosed as are methods for treating a disease condition comprisingadministration of an inhibitory effective amount of purified BH3Interacting domain Death agonist antisense polynucleotide (Korsmeyer,1998).

[0011] Disclosed in US Patent 5,998,583 are BH3 Interacting domain Deathagonist polypeptide and nucleotide derivatives and compositions and usesthereof (Korsmeyer, 1999). There remains, however, a long felt need foradditional agents capable of effectively inhibiting BH3 Interactingdomain Death agonist function.

[0012] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of BH3 Interacting domain Deathagonist expression.

[0013] The present invention provides compositions and methods formodulating BH3 Interacting domain Death agonist expression, includingmodulation of the cleavable form of BH3 Interacting domain Deathagonist, p15 BID.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding BH3 Interacting domain Death agonist, and which modulate theexpression of BH3 Interacting domain Death agonist. Pharmaceutical andother compositions comprising the compounds of the invention are alsoprovided. Further provided are methods of modulating the expression ofBH3 Interacting domain Death agonist in cells or tissues comprisingcontacting said cells or tissues with one or more of the antisensecompounds or compositions of the invention. Further provided are methodsof treating an animal, particularly a human, suspected of having orbeing prone to a disease or condition associated with expression of BH3Interacting domain Death agonist by administering a therapeutically orprophylactically effective amount of one or more of the antisensecompounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding BH3 Interacting domain Death agonist,ultimately modulating the amount of BH3 Interacting domain Death agonistproduced. This is accomplished by providing antisense compounds whichspecifically hybridize with one or more nucleic acids encoding BH3Interacting domain Death agonist. As used herein, the terms “targetnucleic acid” and “nucleic acid encoding BH3 Interacting domain Deathagonist” encompass DNA encoding BH3 Interacting domain Death agonist,RNA (including pre-mRNA and mRNA) transcribed from such DNA, and alsocDNA derived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity which may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression of BH3Interacting domain Death agonist. In the context of the presentinvention, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene. In the context of thepresent invention, inhibition is the preferred form of modulation ofgene expression and mRNA is a preferred target.

[0016] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding BH3 Interacting domain Death agonist. The targetingprocess also includes determination of a site or sites within this genefor the antisense interaction to occur such that the desired effect,e.g., detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the gene. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding BH3 Interacting domain Death agonist, regardless ofthe sequence(s) of such codons.

[0017] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0018] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0019] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0020] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0021] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds.

[0022] “Complementary,” as used herein, refers to the capacity forprecise pairing between two nucleotides. For example, if a nucleotide ata certain position of an oligonucleotide is capable of hydrogen bondingwith a nucleotide at the same position of a DNA or RNA molecule, thenthe oligonucleotide and the DNA or RNA are considered to becomplementary to each other at that position. The oligonucleotide andthe DNA or RNA are complementary to each other when a sufficient numberof corresponding positions in each molecule are occupied by nucleotideswhich can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity or precise pairing such that stableand specific binding occurs between the oligonucleotide and the DNA orRNA target. It is understood in the art that the sequence of anantisense compound need not be 100% complementary to that of its targetnucleic acid to be specifically hybridizable. An antisense compound isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed.

[0023] Antisense and other compounds of the invention which hybridize tothe target and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites.

[0024] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use. Thespecificity and sensitivity of antisense is also harnessed by those ofskill in the art for therapeutic uses. Antisense oligonucleotides havebeen employed as therapeutic moieties in the treatment of disease statesin animals and man. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat oligonucleotides can be useful therapeutic modalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

[0025] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0026] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about50 nucleobases (i.e., from about 8 to about 50 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 30 nucleobases. Antisense compounds include ribozymes, externalguide sequence (EGS) oligonucleotides (oligozymes), and other shortcatalytic RNAs or catalytic oligonucleotides which hybridize to thetarget nucleic acid and modulate its expression.

[0027] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

[0028] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0029] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage, i.e., a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0030] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697; and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0031] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0032] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269; and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0033] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0034] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0035] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, O₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylamino-ethoxyethoxy (also known in the art as2′-O-dimethylamino-ethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0036] A further prefered modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—), group bridging the 2′ oxygenatom and the 3′ or 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0037] Other preferred modifications include 2¹-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂-CH═CH₂),2′-O-allkyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modificationmay be in the arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0038] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example, 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0039] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

[0040] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992,the entire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937). Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999), which isincorporated herein by reference in its entirety.

[0041] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0042] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0043] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0044] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,CA). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0045] The antisense compounds of the invention are synthesized in vitroand do not include antisense compositions of biological origin, orgenetic vector constructs designed to direct the in vivo synthesis ofantisense molecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0046] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0047] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993, orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0048] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0049] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfoic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0050] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example, hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0051] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of BH3 Interacting domain Death agonist is treated byadministering antisense compounds in accordance with this invention. Thecompounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of an antisense compound to asuitable pharmaceutically acceptable diluent or carrier. Use of theantisense compounds and methods of the invention may also be usefulprophylactically, e.g., to prevent or delay infection, inflammation ortumor formation, for example.

[0052] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding BH3 Interacting domain Death agonist, enabling sandwich andother assays to easily be constructed to exploit this fact.Hybridization of the antisense oligonucleotides of the invention with anucleic acid encoding BH3 Interacting domain Death agonist can bedetected by means known in the art. Such means may include conjugationof an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of BH3 Interacting domain Deathagonist in a sample may also be prepared.

[0053] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0054] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.,dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999, which is incorporated herein byreference in its entirety.

[0055] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Prefered bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Preferedfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). Also prefered are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly prefered combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids;

[0056] polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999), each of which is incorporated herein by referencein their entirety.

[0057] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0058] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0059] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0060] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0061] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0062] Emulsions

[0063] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

[0064] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0065] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0066] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0067] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0068] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0069] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0070] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of reasons of ease of formulation,efficacy from an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0071] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0072] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0073] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0074] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0075] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0076] Liposomes

[0077] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0078] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0079] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0080] Further advantages of liposomes include: liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; and liposomescan protect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0081] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranes.As the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0082] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0083] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0084] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0085] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0086] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0087] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0088] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0089] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765). Various liposomes comprising one or more glycolipids areknown in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987,507, 64) reported the ability of monosialoganglioside G_(M1),galactocerebroside sulfate and phosphatidylinositol to improve bloodhalf-lives of liposomes. These findings were expounded upon by Gabizonet al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No.4,837,028 and WO 88/04924, both to Allen et al., disclose liposomescomprising (1) sphingomyelin and (2) the ganglioside G_(M1) or agalactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.)discloses liposomes comprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0090] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0091] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0092] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0093] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0094] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0095] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0096] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0097] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0098] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0099] Penetration Enhancers

[0100] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0101] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0102] Surfactants:

[0103] In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

[0104] Fatty Acids:

[0105] Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0106] Bile Salts:

[0107] The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydrofusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0108] Chelating Agents:

[0109] Chelating agents, as used in connection with the presentinvention, can be defined as compounds that remove metallic ions fromsolution by forming complexes therewith, with the result that absorptionof oligonucleotides through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0110] Non-chelating Non-surfactants:

[0111] As used herein, non-chelating non-surfactant penetrationenhancing compounds can be defined as compounds that demonstrateinsignificant activity as chelating agents or as surfactants but thatnonetheless enhance absorption of oligonucleotides through thealimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

[0112] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al., U.S. Pat. No. 5,705,188), cationic glycerolderivatives, and polycationic molecules, such as polylysine (Lollo etal., PCT Application WO 97/30731), are also known to enhance thecellular uptake of oligonucleotides.

[0113] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0114] Carriers

[0115] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0116] Excipients

[0117] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0118] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0119] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0120] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0121] Other Components

[0122] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0123] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0124] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N. J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0125] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0126] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0127] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0128] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxyand 2′-alkoxy Amidites

[0129] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g.,Chemgenes, Needham Mass. or Glen Research, Inc., Sterling, Va.). Other2′-O-alkoxy substituted nucleoside amidites are prepared as described inU.S. Pat. No. 5,506,351, herein incorporated by reference. Foroligonucleotides synthesized using 2′-alkoxy amidites, the standardcycle for unmodified oligonucleotides was utilized, except the wait stepafter pulse delivery of tetrazole and base was increased to 360 seconds.

[0130] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me—C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

[0131] 2′-Fluoro Amidites

[0132] 2′-Fluorodeoxyadenosine Amidites

[0133] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et al., J. Med. Chem., 1993, 36, 831-841] and U.S.Pat. No. 5,670,633, herein incorporated by reference. Briefly, theprotected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine wassynthesized utilizing commercially available9-beta-D-arabinofuranosyladenine as starting material and by modifyingliterature procedures whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-trityl group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0134] 2′-Fluorodeoxyguanosine

[0135] The synthesis of 2¹-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0136] 2′-Fluorouridine

[0137] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0138] 2′-Fluorodeoxycytidine

[0139] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0140] 2′-O-(2-Methoxyethyl) Modified Amidites

[0141] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

[0142] 2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0143] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 h) to give a solid that was crushed to a light tan powder(57 g, 85% crude yield). The NMR spectrum was consistent with thestructure, contaminated with phenol as its sodium salt (ca. 5%). Thematerial was used as is for further reactions (or it can be purifiedfurther by column chromatography using a gradient of methanol in ethylacetate (10-25%) to give a white solid, mp 222-4° C.).

[0144] 2′-O-Methoxyethyl-5-methyluridine

[0145] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. Additional material was obtained byreworking impure fractions.

[0146] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0147] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0148]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0149] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by TLC by first quenching the TLC sample with the additionof MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

[0150]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0151] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to -5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

[0152] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0153] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄0H (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2x200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0154]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0155] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, TLC showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 9). The residue was chromatographed ona 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0156]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0157]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) wereadded with stirring, under a nitrogen atmosphere. The resulting mixturewas stirred for 20 hours at room temperature (TLC showed the reaction tobe 95% complete). The reaction mixture was extracted with saturatedNaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

[0158] 2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0159] 2′-(Dimethylaminooxyethoxy) NMucleoside Amidites

[0160] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0161] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0162] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0g, 0.416 mmol), dimethylaminopyridine (0.66g, 0.013eq, 0.0054mmol)were dissolved in dry pyridine (500 ml) at ambient temperature under anargon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

[0163]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0164] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

[0165]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0166]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂0₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

[0167]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0168]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

[0169]5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0170]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 100C under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO3 solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6g, 80%).

[0171] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0172] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

[0173] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0174] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

[0175]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0176] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL).

[0177] To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67mmol) was added and dried over P₂O₅ under high vacuum overnight at 40°C. Then the reaction mixture was dissolved in anhydrous acetonitrile(8.4 mL) and 2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12mL, 6.08 mmol) was added. The reaction mixture was stirred at ambienttemperature for 4 hrs under inert atmosphere. The progress of thereaction was monitored by TLC (hexane:ethyl acetate 1:1). The solventwas evaporated, then the residue was dissolved in ethyl acetate (70 mL)and washed with 5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was driedover anhydrous Na₂SO₄ and concentrated. Residue obtained waschromatographed (ethyl acetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

[0178] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0179] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0180]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0181] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0182] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0183] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O-CH₂-O-CH₂-N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0184] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

[0185] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) isslowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-, 2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

[0186]5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine

[0187] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour.

[0188] The reaction mixture is poured into water (200 mL) and extractedwith CH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers are washed withsaturated NaHCO₃ solution, followed by saturated NaCl solution and driedover anhydrous sodium sulfate. Evaporation of the solvent followed bysilica gel chromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1%triethylamine) gives the title compound.

[0189]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramiditeDiisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2

[0190] Oligonucleotide Synthesis

[0191] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using standard phosphoramidite chemistrywith oxidation by iodine.

[0192] Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0193] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0194] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0195] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0196] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0197] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0198] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0199] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3

[0200] Oligonucleoside Synthesis

[0201] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P=O or P=S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0202] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0203] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0204] PNA Synthesis

[0205] Peptide nucleic acids (PNAS) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0206] Synthesis of Chimeric Oligonucleotides

[0207] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 31 or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0208] [2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0209] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to 1/2 volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[0210] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[0211] [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0212] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0213] [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0214] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0215] Oligonucleotide Isolation

[0216] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹p nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0217] oligonucleotide Synthesis—96 Well Plate Format

[0218] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

[0219] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0220] Oligonucleotide Analysis—96 Well Plate Format

[0221] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0222] Cell Culture and oligonucleotide Treatment

[0223] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,Ribonuclease protection assays, or RT-PCR.

[0224] T-24 Cells:

[0225] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, MD) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0226] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0227] A549 Cells:

[0228] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

[0229] NHDF Cells:

[0230] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0231] HEK Cells:

[0232] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville Md.). HEKs were routinely maintainedin Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

[0233] b.END Cells:

[0234] The mouse brain endothelial cell line b.END was obtained from Dr.Werner Risau at the Max Plank Instititute (Bad Nauheim, Germany). b.ENDcells were routinely cultured in DMEM, high glucose (Gibco/LifeTechnologies, Gaithersburg, MD) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 3000 cells/well for use in RT-PCR analysis.

[0235] For Northern blotting or other analyses, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0236] Treatment with Antisense Compounds:

[0237] When cells reached 80% conf luency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™ -1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™ -1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

[0238] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell 35 line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10

[0239] Analysis of Oligonucleotide Inhibition of BH3 Interacting DomainDeath Agonist Expression

[0240] Antisense modulation of BH3 Interacting domain Death agonistexpression can be assayed in a variety of ways known in the art. Forexample, BH3 Interacting domain Death agonist mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+mRNA. Methods of RNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons,Inc., 1993. Northern blot analysis is routine in the art and is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

[0241] Protein levels of BH3 Interacting domain Death agonist can bequantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting), ELISA orfluorescence-activated cell sorting (FACS). Antibodies directed to BH3Interacting domain Death agonist can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0242] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998. Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

[0243] Poly(A)+ mRNA Isolation

[0244] Poly(A)+ mRNA was isolated according to Miura et al., Clin.Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60AL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine, Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C. was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0245] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0246] Total RNA Isolation

[0247] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 100 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 100 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPEwas then added to each well of the RNEASY 96™ plate and the vacuumapplied for a period of 15 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 10 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 60 μL water into each well, incubating1 minute, and then applying the vacuum for 30 seconds. The elution stepwas repeated with an additional 60 μL water.

[0248] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia, Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0249] Real-time Quantitative PCR Analysis of BH3 Interacting DomainDeath Agonist mRNA Levels

[0250] Quantitation of BH3 Interacting domain Death agonist mRNA levelswas determined by real-time quantitative PCR using the ABI PRISM™ 7700Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR, in which amplificationproducts are quantitated after the PCR is completed, products inreal-time quantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., JOE, FAM, orVIC, obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end ofthe probe and a quencher dye (e.g., TAMRA, obtained from either OperonTechnologies Inc., Alameda, CA or PE-Applied Biosystems, Foster City,CA) is attached to the 3′ end of the probe. When the probe and dyes areintact, reporter dye emission is quenched by the proximity of the 3′quencher dye. During amplification, annealing of the probe to the targetsequence creates a substrate that can be cleaved by the 5′-exonucleaseactivity of Taq polymerase. During the extension phase of the PCRamplification cycle, cleavage of the probe by Taq polymerase releasesthe reporter dye from the remainder of the probe (and hence from thequencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™7700 Sequence Detection System. In each assay, a series of parallelreactions containing serial dilutions of mRNA from untreated controlsamples generates a standard curve that is used to quantitate thepercent inhibition after antisense oligonucleotide treatment of testsamples.

[0251] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0252] PCR reagents were obtained from PE-Applied Biosystems, FosterCity, Calif. RT-PCR reactions were carried out by adding 25 μL PCRcocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTPand dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer,and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MULV reverse transcriptase) to 96 well plates containing 25 μLtotal RNA solution. The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

[0253] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al.,Analytical Biochemistry, 1998, 265, 368-374.

[0254] In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 25 uL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0255] Probes and primers to human BH3 Interacting domain Death agonistwere designed to hybridize to a human BH3 Interacting domain Deathagonist sequence, using published sequence information (GenBankaccession number NM_(—)001196.1, incorporated herein as SEQ ID NO: 3).For human BH3 Interacting domain Death agonist the PCR primers were:

[0256] forward primer: AGAAGACATCATCCGGAATATTGC (SEQ ID NO: 4)

[0257] reverse primer: GGAGGGATGCTACGGTCCAT (SEQ ID NO: 5)

[0258] and the PCR probe was: FAM-AGGCACCTCGCCCAGGTCGG-TAMRA (SEQ ID NO:6)

[0259] where FAM (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye. For human GAPDH the PCR primers were:

[0260] forward primer: CAACGGATTTGGTCGTATTGG (SEQ ID NO: 7)

[0261] reverse primer: GGCAACAATATCCACTTTACCAGAGT (SEQ TD NO: 8)

[0262] and the PCR probe was: 5′ 5′ JOE-CGCCTGGTCACCAGGGCTGCT-TAMRA 3′(SEQ ID NO: 9)

[0263] where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

[0264] Probes and primers to mouse BH3 Interacting domain Death agonistwere designed to hybridize to a mouse BH3 Interacting domain Deathagonist sequence, using published sequence information (GenBankaccession number U75506, incorporated herein as SEQ ID NO: 10). Formouse BH3 Interacting domain Death agonist the PCR primers were:

[0265] forward primer: TCGAAGACGAGCTGCAGACA (SEQ ID NO: 11)

[0266] reverse primer: TGGCTCTATTCTTCCTTGGTTGA (SEQ ID NO: 12)

[0267] and the PCR probe was: FAM-CAGCCAGGCCAGCCGCTCC-TAMRA (SEQ ID NO:13)

[0268] where FAM (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMPA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye. For mouse GAPDH the PCR primers were:

[0269] Forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14);

[0270] Reverse primer: GGGTCTCGCTCCTGGAAGCT (SEQ ID NO: 15),

[0271] and the PCR probe was: 5′ 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-(SEQ ID TAMRA 3′ NO: 16)

[0272] where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

Example 14

[0273] Northern Blot Analysis of BH3 Interacting Domain Death AgonistmRNA Levels

[0274] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0275] To detect human BH3 Interacting domain Death agonist, a human BH3Interacting domain Death agonist specific probe was prepared by PCRusing the forward primer AGAAGACATCATCCGGAATATTGC (SEQ ID NO: 4)

[0276] and the reverse primer GGAGGGATGCTACGGTCCAT (SEQ ID NO: 5).

[0277] To normalize for variations in loading and transfer efficiencymembranes were stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0278] To detect mouse BH3 Interacting domain Death agonist, a mouse BH3Interacting domain Death agonist specific probe was prepared by PCRusing the forward primer TCGAAGACGAGCTGCAGACA (SEQ ID NO: 11)

[0279] and the reverse primer TGGCTCTATTCTTCCTTGGTTGA (SEQ ID NO: 12)

[0280] To normalize for variations in loading and transfer efficiencymembranes were stripped and probed for mouse glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0281] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, CA). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0282] Antisense Inhibition of Human BH3 Interacting Domain DeathAgonist Expression by Chimeric Phosphorothioate Oligonucleotides Having2′-MOE Wings and a Deoxy Gap

[0283] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanBH3 Interacting domain Death agonist RNA, using published sequences(GenBank accession number NM_(—)001196.1, incorporated herein as SEQ IDNO: 3, and rseidues 12001-28000 of GenBank accession number AC006285,incorporated herein as SEQ ID NO: 17). The oligonucleotides are shown inTable 1. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 31directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P=S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human BH3 Interacting domain Death agonist mRNA levelsby quantitative real-time PCR as described in other examples herein.Data are averages from two experiments. If present, “N.D.” indicates “nodata”. TABLE 1 Inhibition of human BH3 Interacting domain Death agonistmRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOEwings and a deoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITESEQUENCE % INHIB NO 119845 Coding 3 354 ctttcagaatctgcctctat 67 18119846 Coding 3 707 agtccatcccatttctggct 74 19 119847 5′UTR 17 60actgtggtgagtctcccacc 88 20 119848 5′UTR 17 2083 agtgtcccagtggcgacctg 9021 119849 Coding 17 2134 cacagtccatggcctgggca 98 22 119850 Intron 173582 ctccgcttcctcactccgaa 84 23 119851 Intron 17 3845tactcgggaggctgaggcag 88 24 119852 Intron 17 3906 ccgtctttactaagatacaa 9025 119853 Intron 17 4540 tcaagacagtaaatcctgca 93 26 119854 Intron 174580 ctttttagatcacaggaaaa 89 27 119855 Intron 17 4987gccatttaattccaagaata 92 28 119856 Intron 17 5092 ggcccactgagtggacagct 9329 119857 Intron 17 5373 gcatctgttgtttaaagcca 81 30 119858 Intron 175778 acggagcagccgcatggcac 85 31 119859 Intron 17 6999ggtttcaccatgttggtcag 85 32 119860 Intron 17 7125 tctcggctcactacaacctc 7533 119861 Intron 17 7369 agggacgctgagatctgcgc 92 34 119862 Intron 178083 ggtctcaacaggcagaggca 83 35 119863 Coding 17 8254atccctgaggctggaaccgt 96 36 119864 Coding 17 8282 caaacaccagtaggtttgtg 9237 119865 Coding 17 8287 gaagccaaacaccagtaggt 86 38 119866 Coding 178318 tgcggaagctgttgtcagaa 81 39 119867 Coding 17 8362gggagccagcactggcagct 79 40 119868 Coding 17 8418 cgggagtggctgctgcggtt 8841 119869 Intron 17 9135 gctggacctgggtttcctca 86 42 119870 Intron 179353 aagcagccccttggcaaagg 94 43 119871 Intron 17 9424agggctggatctggaagtgg 74 44 119872 Intron 17 9797 agaaggcagagacattctca 9345 119873 Intron 17 9875 gcccttcctggaccttccca 95 46 119874 Intron 179992 ctcagtctagaggcaaaggc 90 47 119875 Intron 17 10172ctgatccgtctgtgtccagc 96 48 119876 Intron 17 10643 aagtagctgggattacaggc83 49 119877 Intron 17 11311 ggccctgtacctagctccca 94 50 119878 Intron 1711394 atcataccactacactccag 18 51 119879 Intron 17 11641ttgtattttaagtagagacg 85 52 119880 Intron 17 12649 acaaggccagcccccactgg74 53 119881 Intron 17 12734 ggcagagacagagcagactc 77 54 119882 Coding 1712795 tgcctggcaatattccggat 95 55 119883 Coding 17 12811cccgacctgggcgaggtgcc 99 56 119884 Coding 17 12832 gatgctacggtccatgctgt97 57 119885 Coding 17 12894 acctcctccgaccggctggt 98 58 119886 Coding 1714042 ccagggcagtggccaggtcc 95 59 119887 Coding 17 14067ctagggtaggcctgcagcag 94 60 119888 Coding 17 14072 tgtctctagggtaggcctgc94 61 119889 Coding 17 14151 cggagcaaggacggcgtgtg 97 62 119890 Coding 1714178 aaattcactgttgtgtgaaa 96 63 119891 Coding 17 14198tgcgtaggttctggttaata 98 64 119892 Intron 17 14635 agagcagtgggatcacaggc80 65 119893 Intron 17 14694 tgttggccagggtggtctgg 77 66 119894 Intron 1716361 agctgtccatacagactgct 90 67 119895 Coding 17 16678cttctggaactgtccgttca 96 68 119896 3′UTR 17 16753 gttgacatgccagggctccg 9869 119897 3′UTR 17 16798 atagaagtcacagctatctt 95 70 119898 3′UTR 1716933 tgtagatttacagatgtgca 68 71 119899 3′UTR 17 17176ttaagatagatagtccctat 89 72 119900 3′UTR 17 17185 tccttagtattaagatagat 8473 119901 3′UTR 17 17236 tagttcagaatctctgtgcc 62 74 119902 3′UTR 1717267 ccggacttcccatcatttga 86 75 119903 3′UTR 17 17293aaaagtcaagcccctgtgta 77 76 119904 3′UTR 17 17300 aagttgaaaaagtcaagccc 5977 119905 3′UTR 17 17391 gtaaacaaacagtggctgac 82 78 119906 3′UTR 1717415 gtatgcagttagttacctga 86 79 119907 3′UTR 17 17439tgatgtcatggaaagagaaa 80 80 119908 3′UTR 17 17452 tttagcaaagtcttgatgtc 7281 119909 3′UTR 17 17456 tgtctttagcaaagtcttga 89 82 119910 3′UTR 1717588 aacctgttctctccagatgc 80 83 119911 3′UTR 17 17592tagaaacctgttctctccag 85 84 119912 3′UTR 17 17596 tgcttagaaacctgttctct 9085 119913 3′UTR 17 17632 aatttttaaaaagtccaact 24 86 119914 3′UTR 1717731 tgttgcactgtttctaaagc 85 87 119915 3′UTR 17 17757agcttaccactggaacagca 94 88 119916 3′UTR 17 17764 gggacatagcttaccactgg 7089 119917 3′UTR 17 17779 tttaaactgattcctgggac 89 90 119918 3′UTR 1717802 gacccagcatccactgtcgt 36 91 119919 3′UTR 17 17904gaagaaatcatgagtccgtc 86 92 119920 3′UTR 17 17942 gattttaaactcttaaagaa 2993 119921 3′UTR 17 17966 tagagtttgtttttcctttc 77 94 119922 3′UTR 1717970 aatatagagtttgtttttcc 50 95

[0284] As shown in Table 1, SEQ ID NOs 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 87, 88, 89, 90, 92 and 94 demonstrated at least 50%inhibition of human BH3 Interacting domain Death agonist expression inthis assay and are therefore preferred. The target sites to which thesepreferred sequences are complementary are herein referred to as “activesites” and are therefore preferred sites for targeting by compounds ofthe present invention.

Example 16

[0285] Antisense Inhibition of Mouse BH3 Interacting Domain DeathAgonist Expression by Chimeric Phosphorothioate Oligonucleotides Having2′-MOE Wings and a Deoxy Gap.

[0286] In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mouseBH3 Interacting domain Death agonist RNA, using published sequences(GenBank accession number U75506, incorporated herein as SEQ ID NO: 10,and residues 9000-120000 of GenBank accession number AC006945,incorporated herein as SEQ ID NO: 96). The oligonucleotides are shown inTable 2. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on mouse BH3 Interacting domain Death agonist mRNA levelsby quantitative real-time PCR as described in other examples herein.Data are averages from two experiments. If present, “N.D.” indicates “nodata”. TABLE 2 Inhibition of mouse BH3 Interacting domain Death agonistmRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOEwings and a deoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITESEQUENCE % INHIB NO 119925 Start 10 21 cgttgctgacctcagagtcc 48 97 Codon119926 Coding 10 232 ctttcagaatctggctctat 32 98 119927 5′UTR 96 4669ggcccggcgctctactccac 39 99 119928 5′UTR 96 4699 gctaaggcaaaggtttgcgg 58100 119929 5′UTR 96 5004 cgggtccaccaggaggcctg 42 101 119930 5′UTR 965693 gccatggcaccaggcagtag 71 102 119931 5′UTR 96 6758gccaggcagcgtgcccagaa 74 103 119932 5′UTR 96 7548 cttccccattcatacaccta 61104 119933 5′UTR 96 7977 cacttgacaccaacagagac 58 105 119934 5′UTR 968859 gaagcctgtaatcctggcac 73 106 119935 5′UTR 96 9373gaccatgtcctggccagaaa 83 107 119936 5′UTR 96 9439 gtcagtccagtaagggcttt 61108 119937 5′UTR 96 9698 ttagcttagccacagaggga 80 109 119938 5′UTR 969768 cgcctgtgctctcttcctgc 53 110 119939 5′UTR 96 10495cccatcttctggcctccttg 35 111 119940 5′UTR 96 11230 ctgaaactccaggctcagga76 112 119941 5′UTR 96 12652 ctcatggcagctgcagcagt 66 113 119942 5′UTR 9614187 cttgaaaaggaacaaagtgg 44 114 119943 5′UTR 96 14566tctatacactactcataacc 55 115 119944 5′UTR 96 17953 ccatcacagaggccacttct41 116 119945 5′UTR 96 18196 tccatccctggaacaatgtg 58 117 119946 5′UTR 9619488 cagagctcagctttcttccc 68 118 119947 5′UTR 96 19741agctcacagagtccagggaa 55 119 119948 5′UTR 96 19752 caagcactgccagctcacag59 120 119949 Coding 96 19782 tcagagtccatggcacaagc 61 121 119950 Intron96 20989 ttgccaaacagaagacacca 3 122 119951 Intron 96 21013gcagagaaacaggctgtggt 42 123 119952 Coding 96 21182 gtctgtgatgtgcttggccc63 124 119953 Coding 96 21205 tggagaaagccgaacaccag 57 125 119954 Coding96 21259 acaggcagttcccgacccag 71 126 119955 Coding 96 21282ggtctgcctcccagtaagct 27 127 119956 Coding 96 21306 cgtctgtctgcagctcgtct89 128 119957 Intron 96 21950 cttttctgaatgacttgata 39 129 119958 Intron96 22293 cactgataggaagtgtgtcc 54 130 119959 Intron 96 22835ctcagttgctgtaaacacag 57 131 119960 Intron 96 22883 ccacagcgctctgagcactc73 132 119961 Intron 96 23125 gtcctgaagtatcctgacct 72 133 119962 Intron96 23239 gaaataaactagccagaggg 26 134 119963 Coding 96 24169tttcttcctgactttcagaa 33 135 119964 Coding 96 24201 ttgggcgagatgtctggcaa55 136 119965 Coding 96 24208 cgcctatttgggcgagatgt 51 137 119966 Coding96 24264 gaactgtgcggctagctgtc 62 138 119967 Intron 96 24515cgccacaagagaagactgag 54 139 119968 Intron 96 24877 aatgtgtgtgtctttgacag53 140 119969 Intron 96 25363 ctacatgttatcttcccttc 37 141 119970 Coding96 25705 agggctttggccaggcagtt 43 142 119971 Coding 96 25776acagcattgtcattatcagc 67 143 119972 Coding 96 25814 gagcaaagatggtgcgtgac54 144 119973 Coding 96 25830 tgtggaagacatcacggagc 78 145 119974 Coding96 25838 gacagtcgtgtggaagacat 48 146 119975 Coding 96 25858aggttctggttaataaagtt 34 147 119976 Intron 96 26838 gtcattttccagcagtctca77 148 119977 Coding 96 27236 gcgggctcctcagtccatct 74 149 119978 3′UTR96 27315 gttctctgggacctgtcttc 44 150 119979 3′UTR 96 27474tcattcccaagtgggaaccc 49 151 119980 3′UTR 96 27577 cagaagcccacctacatggt44 152 119981 3′UTR 96 27608 atgcacctctcctaatgctg 58 153 119982 3′UTR 9627612 gccgatgcacctctcctaat 67 154 119983 3′UTR 96 27657gagcacttcagtgtccacta 56 155 119984 3′UTR 96 27700 agatcagccattcggctttt58 156 119985 3′UTR 96 27711 cccatggtttgagatcagcc 75 157 119986 3′UTR 9627788 gatagaaatcttgagataat 11 158 119987 3′UTR 96 27834caccacacagataagtcgtg 65 159 119988 3′UTR 96 27842 gtaactgacaccacacagat60 160 119989 3′UTR 96 27851 agcctgagtgtaactgacac 54 161 119990 3′UTR 9627859 gtagcaagagcctgagtgta 48 162 119991 3′UTR 96 27868ttgcattccgtagcaagagc 51 163 119992 3′UTR 96 27934 agtgacctgctgctgtttta37 164 119993 3′UTR 96 28042 cttttgatatggaatcttct 50 165 119994 3′UTR 9628067 aatacagaagcggagggaac 32 166 119995 3′UTR 96 28083gaggccttgtctctgaaata 78 167 119996 3′UTR 96 28107 cgtaacaacgcttgaggata63 168 119997 3′UTR 96 28145 gctgacgatcccagctttaa 38 169 119998 3′UTR 9628167 cttgcaggctgtggcggctc 65 170 119999 3′UTR 96 28170atacttgcaggctgtggcgg 52 171 120000 3′UTR 96 28192 ctgggatgagttcagaacta73 172 120001 3′UTR 96 28332 cacatatttttagaacagaa 38 173 120002 3′UTR 9628378 gagccttttattttgaagaa 60 174

[0287] As shown in Table 2, SEQ ID NOs 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 123, 124, 125, 126, 128, 129, 130, 131, 132, 133,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173 and 174 demonstrated atleast 30% inhibition of mouse BH3 Interacting domain eath agonistexpression in this experiment and are therefore preferred. The targetsites to which these preferred sequences are complementary are hereinreferred to as “active sites” and are therefore preferred sites fortargeting by compounds of the present invention.

Example 17

[0288] Western Blot Analysis of BH3 Interacting Domain Death AgonistProtein Levels

[0289] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to BH3 Interactingdomain Death agonist is used, with a radiolabelled or fluorescentlylabeled secondary antibody directed against the primary antibodyspecies. Bands are visualized using a PHOSPHORIMAGER™ (MolecularDynamics, Sunnyvale, Calif.).

Example 18

[0290] Effect of BH3 Interacting Death Domain Antisense Oligonucleotidesin a Fas Cross-linking Antibody Murine Model for Hepatitis

[0291] Injection of agonistic Fas-specific antibody into mice can inducemassive hepatocyte apoptosis and liver hemorrhage, and death from acutehepatic failure (Ogasawara, J., et al., Nature, 1993, 364, 806-809).Apoptosis-mediated aberrant cell death has been shown to play animportant role in a number of human diseases. For example, in hepatitis,Fas and Fas ligand up-regulated expression are correlated with liverdamage and apoptosis. It is thought that apoptosis in the livers ofpatients with fulminant hepatitis, acute and chronic viral hepatitis orautoimmune hepatitis, as well as chemical or drug induced liverintoxication may result from Fas activation on hepatocytes. There arevarious indices of liver damage and/or apoptosis that are commonly used.These include measurement of the liver enzymes, AST and ALT.

[0292] Eight to ten week-old female Balb/c mice were intraperitoneallyinjected with oligonucleotides 119935 (SEQ ID NO. 107) at 24 mg/kg,daily for 4 days or with saline at a dose of 7 ug. Four hours after thelast dose, 7.5 ug of mouse Fas antibody (Pharmingen, San Diego, Calif.)was injected into the mice. Mortality of the mice was measured for 48hours following antibody treatment.

[0293] Results are shown in Table 3. Mortality is expressed as percentsurvival. TABLE 3 Protective Effects of BH3 Interacting Death DomainAntisense Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides inFas Antibody Cross-linking Induced Death in Balb/c Mice SEQ ID PercentSurvival ISIS # NO: 4 Hr 6 Hr 8 Hr 12 Hr 24 Hr 48 Saline — 100  90  20 0  0  0 119935 107 100 100 100 100 100 100

[0294] Oligonucleotides 119935 (SEQ ID NO. 107) completely protected theFas-antibody treated mice from death. Injection with saline alone didnot confer any protective effect.

[0295] After challenge with a higher dose of Fas antibody (15 ug),protection from fulminant death by the BH3 Interacting Death Domainantisense oligonucleotides was lost with survival rates dropping to 1percent at 5 hours post-treatment. An increase in antisenseoligonucleotide dosage to 50 mg/kg given 6 times every 3 days alsofailed to produce protection from fulminant death at the higher dose ofFas antibody.

[0296] BH3 Interacting Death Domain antisense oligonucleotides were alsoshown to override sensitization to Fas antibody-induced death by Bcl-xLantisense oligonucleotides in the same model.

[0297] In these experiments, 8-10 week-old female Balb/c mice wereintraperitoneally injected with oligonucleotides ISIS 16009 (SEQ ID NO.175, targeting murine Bcl-xL) alone or in combination with ISIS 119935(SEQ ID NO. 107) at 50 mg/kg, 6 times a day for two days or with salineat a dose of 7 ug. ISIS 16009 is a chimeric oligonucleotide (“gapmer”)20 nucleotides in length, composed of a central “gap” region consistingof ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2¹-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide.Cytidine residues in the “wings” are 5-methylcytidines. Four hours afterthe last dose, 7 ug of mouse Fas antibody (Pharmingen, San Diego,Calif.) was injected into the mice. Mortality of the mice was measuredfor 48 hours following antibody treatment. Results are shown in Table 4.Mortality is expressed as percent survival. N.D. indicates no data forthese timepoints. TABLE 4 Protective Effects of BH3 Interacting DeathDomain Antisense Oligonucleotides in Fas Antibody Cross-linking InducedDeath in Balb/c Mice sensitized by Bcl-xL antisense oligonucletidetreatment. Percent Survival ISIS # SEQ ID 4 Hr 6 Hr 8 Hr 12 24 48 saline—  90  60  20  0  0  0 16009 175  90  30  20  10 N.D. N.D. 119935 + 107100 100 100 100 100 100

[0298] Western blot analysis of Bcl-xL and BH3 Interacting Death Domainproteins revealed that the expression levels of both targets was reducedto greater than 90%.

Example 20

[0299] Effect of BH3 Interacting Death Domain Antisense Oligonucleotidesin an Endotoxin and D(+)-Galactosamine-induced Murine Model of FulminantHepatitis and Liver Injury

[0300] The lipopolysaccharide/D-galactosamine or LPS/GalN model is awell known experimental model of toxin-induced hepatitis. Injection ofthe endotoxin, lipopolysaccharide (LPS), induces septic shock death inthe mouse, though with LPS alone, the mouse liver does not sustain majordamage. Injection of D-Galactosamine (GalN), while metabolized in livercausing depletion of UTP, is not lethal to mice. It does, however,sensitize animals to TNF-α or LPS-induced endotoxic shock by over 1,000fold. In the presence of GalN, LPS induces apoptotic cell death inliver, thymus, spleen, lymph nodes and the kidney and results infulminant death in animals. The liver injury is known to be transferablevia the serum, suggesting a mechanism of action under TNF-α control.Further support for this mechanism is provided by the finding that TNFR1knockout mice are resistant to LPS/GalN-induced liver injury and death.

[0301] Eight-week-old female Balb/c mice were used to assess theactivity of BH3 Interacting Death Domain antisense oligonucleotides inthe endotoxin and D(+)-Galactosamine-induced murine model of fulminanthepatitis and liver injury. Mice were intraperitoneally pretreated with24 mg/kg of ISIS 119935 (SEQ ID NO. 107) four times a day for 2 days.Control mice were injected with saline. One day after the last dose ofoligonucleotide, mice were injected intraperitoneally with 5ng LPS(DIFCO laboratories) and 20mg D-Galactosamine (Sigma) per animal insaline. At time intervals of 5.5, 7.5, 9.5, 21.5, 30, 45 and 53 hoursafter the final dose, animals were monitored for survival rates. Resultsare shown in Table 5. TABLE 5 Protective Effects of BH3 InteractingDeath Domain Antisense Oligonucleotides in Endotoxin-Mediated Death inBalb/c Mice Percent Survival ISIS # SEQ ID 5.5 7.5 9.5 21.5 30 45 53saline — 100 100  20  20  10  10  10 119935 107 100 100 100 100 100 100100

[0302] BH3 Interacting Death Domain antisense oligonucleotides were alsoshown to override sensitization to endotoxin-mediated death by Bcl-xLantisense oligonucleotides in the same model.

[0303] In these experiments, 8-10 week old female Balb/c mice wereintraperitoneally pretreated with 24 mg/kg of ISIS 16009 (SEQ ID NO.175) alone or in combination with ISIS 119935 (SEQ ID NO. 107) fourtimes a day for 2 days. Control mice were injected with saline. One dayafter the last dose of oligonucleotide, mice were injectedintraperitoneally with 5 ng LPS (DIFCO laboratories) and 20mgD-Galactosamine (Sigma) per animal in saline. At time intervals of 6,6.5, 7, 7.5, 9, 9.5 and 22 hours after the final dose, animals weremonitored for survival rates. Results are shown in Table 6. Mortality isexpressed as percent survival. TABLE 6 Protective Effects of BH3Interacting Death Domain Antisense Oligonucleotides inEndotoxin-Mediated Death in Balb/c Mice sensitized by Bcl-xL antisenseoligonucletide treatment. Percent Survival ISIS # SEQ ID 6 Hr 6.5 7 Hr7.5 9 Hr 9.5 22 saline — 100 100 100 100  70  20  10 16009 175 100  80 30  0  0  0  0 119935 + 107 100 100 100 100 100 100 100

[0304]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 175 <210> SEQ ID NO 1<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400>SEQUENCE: 1 tccgtcatcg ctcctcaggg 20 <210> SEQ ID NO 2 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 2atgcattctg cccccaagga 20 <210> SEQ ID NO 3 <211> LENGTH: 1105 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (141)...(728) <400> SEQUENCE: 3 gggcgggtagtcgaccgtgt ccgcgcgcct gggagacgct gcctcggccc ggacgcgccc 60 gcgcccccgcggctggaggg tggtcgccac tgggacactg tgaaccagga gtgagtcgga 120 gctgccgcgctgcccaggcc atg gac tgt gag gtc aac aac ggt tcc agc 170 Met Asp Cys GluVal Asn Asn Gly Ser Ser 1 5 10 ctc agg gat gag tgc atc aca aac cta ctggtg ttt ggc ttc ctc caa 218 Leu Arg Asp Glu Cys Ile Thr Asn Leu Leu ValPhe Gly Phe Leu Gln 15 20 25 agc tgt tct gac aac agc ttc cgc aga gag ctggac gca ctg ggc cac 266 Ser Cys Ser Asp Asn Ser Phe Arg Arg Glu Leu AspAla Leu Gly His 30 35 40 gag ctg cca gtg ctg gct ccc cag tgg gag ggc tacgat gag ctg cag 314 Glu Leu Pro Val Leu Ala Pro Gln Trp Glu Gly Tyr AspGlu Leu Gln 45 50 55 act gat ggc aac cgc agc agc cac tcc cgc ttg gga agaata gag gca 362 Thr Asp Gly Asn Arg Ser Ser His Ser Arg Leu Gly Arg IleGlu Ala 60 65 70 gat tct gaa agt caa gaa gac atc atc cgg aat att gcc aggcac ctc 410 Asp Ser Glu Ser Gln Glu Asp Ile Ile Arg Asn Ile Ala Arg HisLeu 75 80 85 90 gcc cag gtc ggg gac agc atg gac cgt agc atc cct ccg ggcctg gtg 458 Ala Gln Val Gly Asp Ser Met Asp Arg Ser Ile Pro Pro Gly LeuVal 95 100 105 aac ggc ctg gcc ctg cag ctc agg aac acc agc cgg tcg gaggag gac 506 Asn Gly Leu Ala Leu Gln Leu Arg Asn Thr Ser Arg Ser Glu GluAsp 110 115 120 cgg aac agg gac ctg gcc act gcc ctg gag cag ctg ctg caggcc tac 554 Arg Asn Arg Asp Leu Ala Thr Ala Leu Glu Gln Leu Leu Gln AlaTyr 125 130 135 cct aga gac atg gag aag gag aag acc atg ctg gtg ctg gccctg ctg 602 Pro Arg Asp Met Glu Lys Glu Lys Thr Met Leu Val Leu Ala LeuLeu 140 145 150 ctg gcc aag aag gtg gcc agt cac acg ccg tcc ttg ctc cgtgat gtc 650 Leu Ala Lys Lys Val Ala Ser His Thr Pro Ser Leu Leu Arg AspVal 155 160 165 170 ttt cac aca aca gtg aat ttt att aac cag aac cta cgcacc tac gtg 698 Phe His Thr Thr Val Asn Phe Ile Asn Gln Asn Leu Arg ThrTyr Val 175 180 185 agg agc tta gcc aga aat ggg atg gac tga acggacagttccagaagtgt 748 Arg Ser Leu Ala Arg Asn Gly Met Asp 190 195 gactggctaaagcttgatgt ggtcacagct gtatagctgc ttccagtgta gacggagccc 808 tggcatgtcaacagcgttcc tagagaagac aggctggaag atagctgtga cttctatttt 868 aaagacaatgttaaacttat aacccacttt aaaatatcta cattaatata cttgaatgaa 928 aatgtccatttacacgtatt tgaatggcct tcatatcatc cacacatgaa tctgcacatc 988 tgtaaatctacacacggtgc ctttatttcc actgtgcagg ttcccactta aaaattaaat 1048 tggaaagcaggtttcaagga agtagaaaca aaatacaatt tttttggtaa aaaaaaa 1105 <210> SEQ ID NO4 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 4agaagacatc atccggaata ttgc 24 <210> SEQ ID NO 5 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 5 ggagggatgc tacggtccat 20 <210>SEQ ID NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400>SEQUENCE: 6 aggcacctcg cccaggtcgg 20 <210> SEQ ID NO 7 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: PCR Primer <400> SEQUENCE: 7 caacggattt ggtcgtattg g21 <210> SEQ ID NO 8 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer<400> SEQUENCE: 8 ggcaacaata tccactttac cagagt 26 <210> SEQ ID NO 9<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 9cgcctggtca ccagggctgc t 21 <210> SEQ ID NO 10 <211> LENGTH: 791 <212>TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (19)...(606) <400> SEQUENCE: 10 agctacacag cttgtgccatg gac tct gag gtc agc aac ggt tcc ggc ctg 51 Met Asp Ser Glu Val SerAsn Gly Ser Gly Leu 1 5 10 ggg gcc aag cac atc aca gac ctg ctg gtg ttcggc ttt ctc caa agc 99 Gly Ala Lys His Ile Thr Asp Leu Leu Val Phe GlyPhe Leu Gln Ser 15 20 25 tct ggc tgt act cgc caa gag ctg gag gtg ctg ggtcgg gaa ctg cct 147 Ser Gly Cys Thr Arg Gln Glu Leu Glu Val Leu Gly ArgGlu Leu Pro 30 35 40 gtg caa gct tac tgg gag gca gac ctc gaa gac gag ctgcag aca gac 195 Val Gln Ala Tyr Trp Glu Ala Asp Leu Glu Asp Glu Leu GlnThr Asp 45 50 55 ggc agc cag gcc agc cgc tcc ttc aac caa gga aga ata gagcca gat 243 Gly Ser Gln Ala Ser Arg Ser Phe Asn Gln Gly Arg Ile Glu ProAsp 60 65 70 75 tct gaa agt cag gaa gaa atc atc cac aac att gcc aga catctc gcc 291 Ser Glu Ser Gln Glu Glu Ile Ile His Asn Ile Ala Arg His LeuAla 80 85 90 caa ata ggc gat gag atg gac cac aac atc cag ccc aca ctg gtgaga 339 Gln Ile Gly Asp Glu Met Asp His Asn Ile Gln Pro Thr Leu Val Arg95 100 105 cag cta gcc gca cag ttc atg aat ggc agc ctg tcg gag gaa gacaaa 387 Gln Leu Ala Ala Gln Phe Met Asn Gly Ser Leu Ser Glu Glu Asp Lys110 115 120 agg aac tgc ctg gcc aaa gcc ctt gat gag gtg aag aca gcc ttcccc 435 Arg Asn Cys Leu Ala Lys Ala Leu Asp Glu Val Lys Thr Ala Phe Pro125 130 135 aga gac atg gag aac gac aag gcc atg ctg ata atg aca atg ctgttg 483 Arg Asp Met Glu Asn Asp Lys Ala Met Leu Ile Met Thr Met Leu Leu140 145 150 155 gcc aaa aaa gtg gcc agt cac gca cca tct ttg ctc cgt gatgtc ttc 531 Ala Lys Lys Val Ala Ser His Ala Pro Ser Leu Leu Arg Asp ValPhe 160 165 170 cac acg act gtc aac ttt att aac cag aac cta ttc tcc tatgtg agg 579 His Thr Thr Val Asn Phe Ile Asn Gln Asn Leu Phe Ser Tyr ValArg 175 180 185 aac ttg gtt aga aac gag atg gac tga ggagcccgcacaagcccgat 626 Asn Leu Val Arg Asn Glu Met Asp 190 195 ggtgacactgcctccagagg aaccgcgacc atggaaagac cttggcctga agacaggtcc 686 cagagaacagctgtctccct atttccaggt ggtgggaacc ccaagctggt gattcactgg 746 acatctctgcgttcagcttg agtgtatctg aagagtttac gccgg 791 <210> SEQ ID NO 11 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 11tcgaagacga gctgcagaca 20 <210> SEQ ID NO 12 <211> LENGTH: 23 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 12 tggctctatt cttccttggt tga 23<210> SEQ ID NO 13 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe<400> SEQUENCE: 13 cagccaggcc agccgctcc 19 <210> SEQ ID NO 14 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 14ggcaaattca acggcacagt 20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 15 gggtctcgct cctggaagct 20<210> SEQ ID NO 16 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe<400> SEQUENCE: 16 aaggccgaga atgggaagct tgtcatc 27 <210> SEQ ID NO 17<211> LENGTH: 18000 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (2144)...(2155) <221>NAME/KEY: CDS <222> LOCATION: (8247)...(8457) <221> NAME/KEY: CDS <222>LOCATION: (12772)...(12911) <221> NAME/KEY: CDS <222> LOCATION:(14031)...(14243) <221> NAME/KEY: CDS <222> LOCATION: (16669)...(16680)<400> SEQUENCE: 17 cctgggtatc caagtcgccc tggcagagaa acactgcatgagacacggcg ttagggtctg 60 gtgggagact caccacagtg ccaaggtggc tgcagtttgcttgtgacatg ggcgtgtatc 120 tgagtgtgaa ggaagctggt ttttgtgagc tgcctcccgagctcagaggt gacagtgggc 180 actttcccca cagagacccc tgaagttgtt ccttggagaacaaagtggtg aggggcgggg 240 attccagacc ttgaggcaga agctagggtc tggtccactgttctgtggac tgggcagtgg 300 ccctgggagg tgccgtggcc tctgtggcct gtttcctggggtggggtctg tcttgcgctt 360 tgtctcttgt gggtgcagac tccccttcct ctgctgtggagccggcagat ggccccggag 420 ccagatcctg gtgcctccct gtccacatgc agctcagtcatttgctcttg gtcccttcct 480 atgaaatgca cggccacaca cagccagggt ttctcctgggctccccagag ggagagtagg 540 gtgcagcctg caacagtgca gggtccccag gcctgtgtgagcccccaggt ggggaggtgg 600 gtgatgcgca tgtcagtgct acctcctgcc acctcctctctgcctgggca caggctttct 660 cctctgtttg ctttttattt cctatgtatt caggaaccatgtgaaattgc caatgcttgg 720 ttttgtccta caaaatggcc atttcatttg gttcaacctgatattgtgtc tacacacaca 780 cacgcacaca cacacacaca caggcaaata ctttttaaaacaggattatt ctattcacag 840 tgttctgtag aaatttgtgt tcagtctttt tttttttttttgagacggag tctcgctctg 900 tcgcccaggt cggactgcgg actgcagtgg cgcaatctcggctcactgca agctccgctt 960 cccgggttca cgccattctc ctgcctcagc ctcccgagtagctgggacta caggcgcccg 1020 ccaccgcgcc cggctaattt tttgtatttt tagtagagacggggtttcac cttgttagcc 1080 aggatggtct cgatctcctg acctcatgat ccacccgcctcggcctccca aagtgctggg 1140 attacaggcg tgagccaccg cgcccggcct cagtctttttaagacagctt actgtactga 1200 tgccgcacag atcttttttt tttttcgaga cagggtttcactctcgccca ggctggagtg 1260 cagtggtgca atctccgctc actgcagcct ccacctcctgggtgtaagtg atcctcctgc 1320 ttcagccccc caagtagctg ggcccacagg gcttgcatcaccacacctgg ctaattttgt 1380 atttttgtag agatggggtt tcaccatgtt ggccagactggtcattcttt ttgagatgga 1440 gtctcgctct gtcgcccagg ctggagtgca gtggcgtgatctcggcttac tgcaacctct 1500 ccctcccaag ttcatgccat tctcctgcct cagcctcccgagtagctggg actacaggcg 1560 cccgccacca cgcccggcta attttttgta tttttagtagagacagggtt tcaccgcatt 1620 agccagggtg gtctcgatct cctgacctca tgatccacccgcctcggcct cccaaagtgc 1680 tgggattaca ggcatgagct actgcgtcca gccggaagatttaatttttt aattgtcaaa 1740 tccattctct ctctctataa acattttaca ttttatgataataaaataat ttgtgagccc 1800 acggccccgt ttccctgatg cctgaggtct tcctggggcggcatgggagg gctgaattca 1860 ggtgcggggt cggccccagg gcactgagcg cctgggtgagtatctggaat gaggaaaaca 1920 aagcttggct cccgccaagg agaaagaaac tcaggatgcggggctcaggc caggacctcg 1980 gctcagccgc catttctgga gcacaggcca gcttcgtcgtcctcccgagg ggtcctgacc 2040 agggcttccc aggagcggcc gcccactctg tgtgtccctttccaggtcgc cactgggaca 2100 ctgtgaacca ggagtgagtc ggagctgccg cgctgcccaggcc atg gac tgt gag 2155 Met Asp Cys Glu 1 gtcagaggcc agatcccctgcgggtgcctt gtggggggcg gggtcgaggg gtaagggcct 2215 gcgtgtcccc caccacgcatccctgagggc tgaggctgag cccgcctggc ccttaccaca 2275 gctcggcaca gacgaaccccgcccagcccc ttcactgaag caggcgggag ccgggaagtc 2335 ctacctttcc ctgtcctgcgccttcctcgc actccgcttg tggtgcagcc cctccacacc 2395 gcgcctgggg ctaactgcaagggcgagggg gctttgggtt taagaccatt taacagccat 2455 aggctgtggg tcccagcactttgggaggcc aaggcaggag gattccttga ggccaggagg 2515 tcgaggctac agtgagctgtgattgtgcca ctgcactgca gccctgtcca aacaaacacg 2575 aaagagattt aagaagaagaaagggggcat tagataagca cttcatataa ttctctcaac 2635 tgtaaaagca agacaatacttaccttgtct aaccaatgcc attgctatga ggagcaaata 2695 aatcaataaa ggtcaaataaaagtactgta aactgtaagg tgtttcaaaa attttttaac 2755 ccactggatt taaatttcccttcatagctg ggcgaggtgg cttaggcaca taatcccagt 2815 gacttgggag gcagaagcgagaggattgct tgaagccagg agtttgattg agacaaacct 2875 gggcaacata gtaagaccccgtctttataa agataaaagc ggtggagttc tgggagggga 2935 gcccggagcc cccgccttcagcaggacgct ccctggatgc ttccttgtct ctccttccct 2995 ttaaatggtc tggggagagaaaaatcacag cacacgggtg ctctctccca cccgctgcat 3055 cacatcctcc tcccctccctcctgccgaat tctgcagcct ctgggcgcct cacgctgtcc 3115 tggcagcctc tgggaaggcatctgcgaagt ctaatgcctt ggcacttagt gactgtgtcg 3175 cagttcctga gcatggagagcacccggcac ccaggaggtt ctcaagctgc ccctactggg 3235 ggtcctttcc aaaggtggggacggtgtgga tttcagcgtg gtggctggag ggctgaggca 3295 gtggctcgag tttgatgttagttacataaa cagaggagat tgcaggagct cccccggccc 3355 tgatccaggc ttgttgtcagtgtccaaaag accactctgg gtgccactgt cccttcccac 3415 ctgccgctgc tgttccggcttcgcgctctg gcggcctccg caggtagaac accaccgtca 3475 cccgcgcagc gccctgactcgccggaggag gcgcctgccc tcccgcccgc ctctccccgg 3535 ccccctcagt gagggagggtggacgtcgcc actccccttt cttgccttcg gagtgaggaa 3595 gcggaggcag cagtacggcagcccgcccag ggccacagag ctggggtcac agcgaaacac 3655 tccgaaactt tcttttcaattatagggttc agcctttttt cccatcataa ctttaattct 3715 gtgtagatac ttctattttttatttttatt tttttttttg agattgagtc tctgtgtcgc 3775 ccaggctgga gtgcagtggcacgatctccg ctcactgcag gctccgcctc ccgggttcag 3835 gccattctcc tgcctcagcctcccgagtag ctgggactac aggcgcccgc caccacgccc 3895 ggctcatttt ttgtatcttagtaaagacgg ggtttcaccg tgttagccag gatggtctcg 3955 atctcctgac ctcgtgatccgcccgtctgg gcctcccaaa gtgctgggat tacaggcgtg 4015 agccaccgtg cccggccttattattattat ttttttgaga cgcagttttg ctctgtcgcc 4075 caggctggag tgcagtgatgtgatctccgc tcactgccag ctccgcctcc caagttcatg 4135 ccattctcct gcctcagcctctcgagtagc tgggactaca ggcgcccacc accacgcccg 4195 gctaattttt tatattttagtaaagacggg gtttcaccgt gttagccagg atggtctcga 4255 tctcctgacc tcgcgatctgcccgcctcgg cctcccatag tgctgggatt gcaggcgtga 4315 gccaccgcac ctggctaatttttgtatttt tagtagagat ggggtttcac catgttgccc 4375 aggatgttct cgacctcttgacctcatgat ccgcccgcct cggcttccca aagtgctggg 4435 attacaggcg tgagccaccgcgcccggcca gcaccatctt ttcctttcca ctggaactga 4495 tcttattatt tttgcctccattagatcatt tttgtaacat gtcttgcagg atttactgtc 4555 ttgatcgttt ctcttaacatatttttttcc tgtgatctaa aaagataaaa aactatcaat 4615 tcttttatca aaagtggatctagaggctgg gcatggtggc tcacgccagt aatcccagca 4675 ctttgggagg ccaaggtgggcagatcacct gaggtcaaga gctccagacc agcctggcca 4735 acatggtgaa gccccatctttactaaaaat acaaaaatta gccaggcgtg gtggcacgtg 4795 cctgtaaccc cagctacttgggaggctgag gcaggagaat ccattgaacc tgggaggcag 4855 aggttgcagt gagctgagatggcaccattg tactccagcc tgggcaacag aatgagactc 4915 tgtctccaaa aacaaagtggatctagaaga tcaaaaaagg gcatgattcc atattggcac 4975 agcacaagcc ctattcttggaattaaatgg catccatctt ccgagcccac tcctgtcctg 5035 cagggccggc ccagcctgtccctgaggcac tggtccagac aggagcctgt ccacacagct 5095 gtccactcag tgggcccagtgcttggcttc acggtcactt gcggcaccta gacctcctct 5155 ggcaggtgcc attctttcctctccctccct gccgcctcga gtctttattt tctgtgggat 5215 cttgagtttg ataacctgacctgctgtggt ggcagcaccg ctctgtgtcc agattctgga 5275 tgccaattta ccaagcgcaggtcaaaaaga agtccttggg cagcggctgc ctgcgttagc 5335 ttcttggggc tgctgtaggcggttccaagc aggagagtgg ctttaaacaa cagatgcgga 5395 tcccctcccg gttctagaggcccaaaggct ggaatcccat gttgcccggc tgcttccttc 5455 tggggcgctc tcctggctcctgtggctgcc tctgtcttca catggcgtcc tctctgtgtg 5515 tctctgctta aatctccctctcctttctct tacaaagaca ccagtcattg gatttagggc 5575 ccaccctaat ccaatatgacctcatcttaa cttgattaca tctgtaaaaa ccttattttc 5635 aaataaggtc acattgacaggtacttgggg ttaggacttg cgcttttctt tttgggtgac 5695 acagcttagc ccagcactaactgtgtcacc aggactgtcg cttgaggcag gaatgaagca 5755 catcctgttt gtaagctgtcttgtgccatg cggctgctcc gtacaagaat tgttaggaat 5815 tgatgcagtg gaattttgcatacagttttt cctctcttca gaaacaactt tggagaagta 5875 aaggctgaat agcaatacacaagcacctta ttttatttta ttttagattc aggggcacgt 5935 gtacatgttt gtcacatgggaatattgtgc actggtgggg actgggcttc cggtatcgca 5995 tggagaggga ctctttctgcgctcccccgc ccccgcctcc ctactgtaaa gtgcccggtg 6055 cctgctctct ccatcttcgtgtccatgggc acccattgtt tagctcccac ttataagtga 6115 gaacagtcag tatttgattttctgtttctg agttagttca cttagggtaa tggcctctag 6175 ctccatccgt gttgctgcagaggacatgat tttattcttt tttatggctg cagcaataca 6235 caagctcctt atttttatttatttatttat ttatttttgt tgtttgtttg tttgttttga 6295 gacggagtct ggctctcgtcccccaggctg gagtgcaatg gcgcgatctc ggctcattgc 6355 aacctccacc tcccgggttcaagcgattct cctgcctcag cctcccaagt agctgggact 6415 acagacgccc gccaccaggcccggctaatt tttgtatttt tagtagagac aaggtttcat 6475 catgttggcc aggctggtctcaaactcctg acttcgtgat ccgcccgcct cggcctccca 6535 aagtgctggg attacaggcgtgagccaccg cgcccggcca agctccttat tttaagcatt 6595 ttttttttct tttttgagacagggtttcac tttgtcaccc aggttggagt gcagtggtgt 6655 gatcatggct cattgcagcctcaaacttct gggctcaagt gaccttcccg cctcagtctc 6715 atgagtagct gggactgcaggtgcatgcca ccttggctaa tttttatttt ttgtagagat 6775 ggggatcttg ttgccaggctggtctcaaat tcctgggctc aaacgatcct cctgcctctg 6835 cctcccagag tgccgggattacaggcatca cctagcaaag cattaaaaca atttgctgct 6895 gggtgcagta ggtcacacctgtaatcccag cactttgaga ggccaaggag ttggggggag 6955 ttggggggcg ggcggatcacgaggtcagga gttcgagacc agcctgacca acatggtgaa 7015 acctcgtctc tactaaaaatacaaaaatta gccgggcgtg gtgatgcaca cctgtaatcc 7075 cagctactca ggaagctgaggcgggagaat catttgaacc caggaagcgg aggttgtagt 7135 gagccgagat cacaccactgcactccagcc tgggtgacag agcgagactc catctcaaaa 7195 caaaaacaaa aacaaaaaaacaatttgccc tgtaagaact gtcctctaaa agtttttggt 7255 ttttctaatg aaaaatattatggacttaga gaatagaaat aaatttctgc ctacacttcc 7315 atcttccctc ccacccttctctggcagccc aggaggtctt tttgtgtgaa tctgcgcaga 7375 tctcagcgtc cctgcccttctttgtgtttt gttctctctt ccaccttagg tctttctctg 7435 gtctgggcac acccagctgcagggctcacc tttgcctgta agaatacagc ccccaaacac 7495 agtcagtacc ccaagaacagtccctgccat ctctggcggc acagatgctg gccaagctgc 7555 agctgccagt gctgcccagggagctggaga gctgccggcc aagagcccag cccctctggg 7615 tagagcagga gccagtgccaccactccctg tgggattcgg attaaggaca cacccaccca 7675 aagtaaacca agcttggccaaaggcaggtg cccagctgtg gtcaccactc cgcagtagtt 7735 actgaaaatc ttccatctgcccaataccct cctgagcccg tgaaggagat gagcggaaag 7795 aggctccgcc tgttggaagcacagccagga aaggtgggct cagattgctg aagcctgcag 7855 gggaacttga agaaagcgtgccagcacagg atggcggatg atgcccgcat gacactcgct 7915 cgcctccccg gaacagcctgtggccttctc acctagtggg aagctcccca gccgcgtgtt 7975 tcaggaggtc cagcagattcctctgcagag gaatcccttt ctgcagagtc ggggctcgct 8035 ccctgccatc tacgggcagtgctgcttaaa gctgtggctg cagaccttgc ctctgcctgt 8095 tgagacctcc tgcagggccctccagcccac agggtccctc agctctctgg gacctgtgag 8155 gctctttggg ccagctgcaactggagctct ttgcaggagg ggcctctggc ctggctgaag 8215 tcccggcttc ctgactcccctttcccctca g gtc aac aac ggt tcc agc ctc 8267 Val Asn Asn Gly Ser SerLeu 5 10 agg gat gag tgc atc aca aac cta ctg gtg ttt ggc ttc ctc caa agc8315 Arg Asp Glu Cys Ile Thr Asn Leu Leu Val Phe Gly Phe Leu Gln Ser 1520 25 tgt tct gac aac agc ttc cgc aga gag ctg gac gca ctg ggc cac gag8363 Cys Ser Asp Asn Ser Phe Arg Arg Glu Leu Asp Ala Leu Gly His Glu 3035 40 ctg cca gtg ctg gct ccc cag tgg gag ggc tac gat gag ctg cag act8411 Leu Pro Val Leu Ala Pro Gln Trp Glu Gly Tyr Asp Glu Leu Gln Thr 4550 55 gat ggc aac cgc agc agc cac tcc cgc ttg gga aga ata gag gca g 8457Asp Gly Asn Arg Ser Ser His Ser Arg Leu Gly Arg Ile Glu Ala 60 65 70gtaggcggcc ggccccacct ccttccccaa agctgggctt ctctgtcgcc agtaacattc 8517agggagcctc agggctggaa gggacccccg ggatcactct gcctctgcag tttcagctgc 8577cacgtacgct ggtatcactt aatcacttga ctggtctcta cttgattccc tccagtgctg 8637ctgaactcac tgcctaccat ttttgggtga ctctgttaga aagttcttcc tttctgttga 8697gacagaatct catgtactgg tcttgagtcc cttgtctgga ccaacataga atggtgtttt 8757tatccaattt tccaaatgtg attctgatac aaagattgca gaccacttgt ctggattata 8817taacccaagg ggttctcaca cttggccttg tatcatttca aggacctgga gctttaaatg 8877ctggtgcctg catctcacct ccagagattc tgattggttg gtctgggcat tgctgggtct 8937gggcaaagcc cccaggtggc actaccggtg cggcccctgc ctccccaagc aggcctggct 8997gactgtccca ttgattgagg cccactggtt tcacagtgac ttttgcactg tctatacctg 9057acatatttcc tttcatacat tatgctccgt gattacctat acaagaacac agaagtattt 9117ggaacctcat ttccaggtga ggaaacccag gtccagcaaa gggtaaatga ctagctccag 9177atcacacagc ttgtggccat gttaccactg ggacatgggg ccaggcccct tcttgaggtg 9237ggcctcagcc gccctcccac tgtagggcac tgactccagg tcaccatggt ttccagactg 9297ttcacctttc ctgttgctga tccctgcact ctcctccagc ctccagctcc actccccttt 9357gccaaggggc tgcttctatg gacaggggct gtcccgagtg gaggctgggg gcgagtggag 9417gctcacccac ttccagatcc agccctgcga cgctggcttt cagtagtgtg cacattggaa 9477ttacacgaga aaccttttcc aaatgcaggc cttgggccct actccagctg cctgcatcag 9537gctgttttag ggcgggagac tgcccagagg attctgacgc aggtagaatc cctgccctga 9597aagcctgcag ggatccccgg accctggtcc aggccttcca agctcaaggg ttgcactgcc 9657ctctggtggc tgtgggggag accaacagct gacccagcct tctgcctccc gcctgtctta 9717gatcaggtgc ttgaggacgt ggctggagtt ccccactaga ccggggtggg ggtgggggtg 9777gggggtgggg ggaggtgtct gagaatgtct ctgccttcta atccagccag catatcttct 9837ggctcgccct gaactgagga gaaaccccag atccctttgg gaaggtccag gaagggcagg 9897agtggacagg cacagctctg ctgtcagcac tgctgtgggg gtgactgtag ccccagtctg 9957ccctggtgtt tttctctcgc tcttctccat gccggccttt gcctctagac tgagaaaccg 10017gggttgactc aagtggcacc tgcaaaagtg atcatggcag ttcacttagc ctgcaggtga 10077cagggactgt gaatctagtc cctggcgagc ctggaaagag gggcaaggta gaggctctgg 10137ctgccggggt ttctttggtg agtccgttca ctcggctgga cacagacgga tcaggaaaga 10197ttcctgttgc tactcggctg gtggccagag ggagagagga cgtgtccgta actgaagcaa 10257ggtggataag cttcgggaac gagcgaggca cagattcggt gctgggggag tgatgaggtg 10317ctggaggagc tgggtgctct gctctgcagg gaatcaggaa aactttgggg ctgcagctcc 10377aattgagctg ggccttgggg gttgggtatg tttggttcct tggaaactgg gaagagggaa 10437tggccatctt ttaagcaaaa gcccagcggc tataaatgct acagtgaggc tgggtgcagt 10497ggttcacgcc tgtaatccca gcactttggg aggccaaggc aggtggatca tgaggtcagg 10557agttcaagac caccctagcc aagatggtga aaccccgtct ctactaaaaa aaatatataa 10617aaattagcca ggcggggtgg cgggtgcctg taatcccagc tacttgggag gctgaggtag 10677agaattgttt gaacccggga agcggaggtt gcagtgagct gagattgtac cactgcactc 10737cagcttgggg aacagagtga gactatgtct tgaaaaaaaa aagaaaaaaa aaagctacag 10797tgagtagttg agtttgccta ggaagcgtgg aagttaagtc agacgtactt tcaggctggg 10857tcatgacttg tcacttaagc agagatgagc acttgagagg ttttgaagag aagtgatgtg 10917gcagccttac tgcatgttcc atggacagac tccagggagg ccgtgaaacc cccagagcac 10977agcttctaag aacgtgccca ctccttagca cgtcacttct cccaaccctg ccctgctctg 11037aggtctgtgc tgtgaaggtg gccgagtaga ctggacggca gggagtgggg ctgtcatcat 11097cagatgagag ctaaggggac ccccaccagg gtggcggcaa tggcagaggg taggcaaaac 11157gcttgtattt gcaacataag gtgagatttg acagctgacc gagggtggga gcagcagcca 11217aaaccaaaaa agccagaggg aagttgcaag cacagaaaaa atagaagatt taatgggaga 11277aataacaata gctggcatct attgaacact tactgggagc taggtacagg gcccattcat 11337tcattcatgc aattaaaact ttttttaaga aacggggtct tgctctgttg cccaggctgg 11397agtgtagtgg tatgatcaca gctcactgca gccttgaatt cctggcctca aggagtcctc 11457ccacctcagc ctcctgtgta gctgggatta taggtacgtg cggtacacct ggctcccttt 11517aaaagttttt tgtagaggca gggcacagtg gctcacacct gtaatcccag cactttggga 11577ggccaaggca ggaggatcac aaggtcagga gttcgagacc agcctgacca acatggtgaa 11637acccgtctct acttaaaata caaaaattag ccgggtgtgg tggcgggcgc ctgtaatccc 11697agctactcag gaggctgaag catgagactt gcttgaaccc aggaggcgaa ggttgcagtg 11757agccgagatc gcgccactgc actccagcct gggtgacaga gcaagactcc gtctcaaaaa 11817aaaaaaaaaa gtttcttgta gaggcagggc cttgctttgt tgctggtgca atcacggctc 11877actgcatcct ctaactcctg gccttaagca atcttctgtc ctcagcctcc caaagcactg 11937ggattacagg catgcatgac cacacctggt ccctgccatt gtttattgag cacctactga 11997gtgccatgta ttaagtgctg ggtatttgtc agtggacaaa acagattaaa aaaatcacag 12057cccttaggga gcttaccttc tggcaggggc gtcagacaat aacacagcaa gtgctgagga 12117agaaacggag gcggcaggga gcgtggcagt tgagcgtggc cttcatggag ctgcgacagt 12177ggtactcggg caggggcagc acggaggctg tgcgccagag gaggaggact gaggggcaag 12237ggggagagct ctggttggaa aggcagggga gattctccag ggccttgccg gtgccagtga 12297caactggggt tttcctgaga cgggactgcg aggaatgggg gctctcaggc ttgagagggc 12357aaaagtgggt ctgggatgcc gtctgcccac agagcccctt ccccaacggc tgcccaggcc 12417aaggccaacc ctgttgggtt gtgtggtgtg agccatgaag ccgctgccag gcttgtacct 12477caggcgtggt cgtgatgccc cagcttcacc ggccctgcct gtggggacgt ggtgcctgtg 12537tgcgggagcc tgggcctcag ccgaggccct gagctccggc actgcccaga acccagctca 12597gcgctggtac tcagcccgcc cgctgtggcc ctggtggagt ggagcacgtg cccagtgggg 12657gctggccttg tcccatcgcg gacctgtcct ttcccggggc agggtggtgt gggagagggt 12717atcagggaca ttttctgagt ctgctctgtc tctgccgccc ctgcctgaac acag at 12773 Asp75 tct gaa agt caa gaa gac atc atc cgg aat att gcc agg cac ctc gcc 12821Ser Glu Ser Gln Glu Asp Ile Ile Arg Asn Ile Ala Arg His Leu Ala 80 85 90cag gtc ggg gac agc atg gac cgt agc atc cct ccg ggc ctg gtg aac 12869Gln Val Gly Asp Ser Met Asp Arg Ser Ile Pro Pro Gly Leu Val Asn 95 100105 ggc ctg gcc ctg cag ctc agg aac acc agc cgg tcg gag gag 12911 GlyLeu Ala Leu Gln Leu Arg Asn Thr Ser Arg Ser Glu Glu 110 115 120gtgagtgagg gcctgaggac cgcgtgggcg ggcaagtgag ccaagggggc ctgtcccctg 12971cctctcacca ggcagcccac tgtcccgtga ggccactcaa ctcgtgactg tcaggtccag 13031aactctgacg aagtaactgg acgtagggta tggttcattg ccttgcagaa gatttcagct 13091ggttgacatc gaggaaacct gaaccttaaa tcagagtaaa gagtttaggg gtaaaagcct 13151ctaaaagatg aacgaagcat gtttggccaa cagaagaaac agacgcttcc tttggttgta 13211gggagtttaa taatggtgcc agtgagaacc gtaagccctg ggagtggtgc ctgctgctct 13271gctgagctcc ttggttggaa tccacacaac tttctgagct ctaccatctg cttggcactg 13331ttggggatac aagattggtc cggggcactg tgtccccaga acacttagcg gaaagaacta 13391catcctccca actgccaaat gcaggcctgt agcggtagga gctgagagga gagaaagttc 13451cactttttcg actctaccag ctgaaaatgc aggcgtcctc acctcctaga aatccaatca 13511tgcttctgtt cagtggggcc agcctgtgat gtcccagcag ctgcctagaa cgcaggagtg 13571gctggcgcac tcccatgtaa ctctgcatgt gcgccgaccg cctgacggtc cttgccagcc 13631ttgtagtctg tctagtgtcc cccaggaacc cccttcctcc tgtccattca gctaggtctg 13691caccaataaa atgggcctaa ggcgtcgcag gtggtcacta gttctggact cgaagtgcct 13751tgggcgcagg gatgacccag gcttcttgta tcccatcacc gtctaacagt gggcacatgg 13811gctcaccaca catgcgtttg cttaccgagc cccctgcagg gagtgattgc agtcttccct 13871ttccattgcc tctcagaact caactgtttc tcattctttc cgcccagcag ccctggatac 13931ttaataagta ctttgaagtg cttcttcata ctggggactg tctttccttt gagagggaag 13991agtattagta aaccaggttc tgtgtgcccc tctgtgcag gac cgg aac agg gac 14045 AspArg Asn Arg Asp 125 ctg gcc act gcc ctg gag cag ctg ctg cag gcc tac cctaga gac atg 14093 Leu Ala Thr Ala Leu Glu Gln Leu Leu Gln Ala Tyr ProArg Asp Met 130 135 140 gag aag gag aag acc atg ctg gtg ctg gcc ctg ctgctg gcc aag aag 14141 Glu Lys Glu Lys Thr Met Leu Val Leu Ala Leu LeuLeu Ala Lys Lys 145 150 155 gtg gcc agt cac acg ccg tcc ttg ctc cgt gatgtc ttt cac aca aca 14189 Val Ala Ser His Thr Pro Ser Leu Leu Arg AspVal Phe His Thr Thr 160 165 170 gtg aat ttt att aac cag aac cta cgc acctac gtg agg agc tta gcc 14237 Val Asn Phe Ile Asn Gln Asn Leu Arg ThrTyr Val Arg Ser Leu Ala 175 180 185 190 aga aat gtaagaaccc ttgaggtcagctccttccct gcctgccgcc catgcccttt 14293 Arg Asn tctctggaag gttgagaagcccagcggggc ccctgcctct gatgccagca caagggttac 14353 aggctgtcct gctcgggtttggttttgctg ttgtgagcta gaaagctgtg tgtaaaggtg 14413 acgaagagca cccagagtcctttggagctt tagcagctta ctattggaga catgctccat 14473 tcagaggggt ggcaaaggctcacgtcacac tcctggtggg gtcctcaagg cacaagcagg 14533 tacagagtgg aaggaaggggctggagggct cacaatgagc ttttcagacc tctcaccttg 14593 ccataaaaaa taagtgtaatgtggccagtg cggtggctca tgcctgtgat cccactgctc 14653 tgggaggcca aggcaggtggatcacctgag gtcaggagtt ccagaccacc ctggccaaca 14713 gggtgaaagc ccgtctctactaaaatacaa aaattagccg ggcatggtgg cgcacacctg 14773 tagtcccagc tactcaggaggctgaggcag gagaactgct tgaaccctgg aggcagaggt 14833 tgcagtgaac tgagatcgcaccactgcact ttagcctggg cgacagagca agactccatc 14893 tcaaaaaaaa ggtgtaatgtgaaccaaaac gagtagtcaa aaaagggggg gaactgtctg 14953 aaatcttttc cagagcacatctgtcccata accaggtatt acaagtcaca gtctaaaggc 15013 tgggcatggt ggctcaagcctgtaatccca gcgatttggg aagcagaagc agtgggattg 15073 cttgaggcca ggagtttgagacaaaactga gcaacatggc gagaccctgt ctctaaaaaa 15133 tttataaaaa taattagctgagggccaggc gcggtggctc acgcctgtaa tcccagcact 15193 ttgggaggcc aaggcaggcggatcatgaag tcaggagttc aagaccagcc tggccaagat 15253 ggtgaaaccc cgtttctactaaaaatacaa aaaaaattag ctgggtgtgg tggcgggcgc 15313 ctgtaatccc agctactcaggaggctaagg caggagaatc gcttgaaccc tggtggcaga 15373 ggttgcagtg agccgcaatcacgccactgc actccagcct ggatgatggg gtaagactgt 15433 ctcaaaaaaa aaaaaaattagctgagcatg gtggcgtacg cctgtagttc acgccgtcat 15493 ggaggttgag gcagctcctcaggaggctgg ggcagaagga tctctttgct tgagcccagg 15553 agttcaaggc tgcagtgagctgattgtgcc actgcactcc agcctgaaca aaaacaagac 15613 ctgtctctaa aaacaaacatacagtgttca caatgctgcc caagaagggc cagtttttgc 15673 agctgccccc atgtagcaaaatctggtgct tctgtttcat agacccaaat ggaaattaag 15733 tggatgtgtc ttatttgtaaatttaaaaat attagcgaat gtttgggaat tttttttttt 15793 tttttttttg agacagaattttgctcttgt tgcccaggct ggagtgcaat ggcacgatct 15853 cagctcacca caacctctgcctcccaggtt caagcgattc tcctgcctca gccccccaag 15913 aagctgggat tacaggcacacaccaccatg accggctaat tttgtatttt tagtagagat 15973 gaggtttctc ccatgttaggctggtctcga actcccaacc tcaggtgatc cgcccacctc 16033 ggcctcccaa agtgctgggattacaggcgt gagccactgc gcccggccta atgtttggga 16093 ttttatgaca tgtcagaagcattacttcag gctttggttt ttaagtaaaa tagcatctaa 16153 tcctctactg agaactcataagaaaacatt ccttatatgc tgtggtcttc agttatacaa 16213 gcattttaaa aacaggagaatgaatataaa tcttaaatca ggcattaaac ccagctgaat 16273 tgttggaagg aggtaagcctgagaccattc ctggacagct tttaccaaca cccatgtaaa 16333 gggggaaagg gtgggcaagacgtgtgcagc agtctgtatg gacagcttac cagagactga 16393 gggctgaggc agaatcgtgattcctctgac ccagcagggg cctcctgaca ccgtcagtgc 16453 cttggagatg tgaatacccacctcaccgcc tgaacggcct gtttttgcag ttgcccccat 16513 gtagcaaaaa gtaggatgcacggataggac ttcaggggtc tggagaacat gtttttgcat 16573 aaaccccagc tttgctctactgtggcacag agctctggag cctggtttgt gaatgagcct 16633 agctgattct ggctttttctcctttcttgc tctag ggg atg gac tga acggacagtt 16690 Gly Met Asp 195ccagaagtgt gactggctaa agctcgatgt ggtcacagct gtatagctgc ttccagtgta 16750gacggagccc tggcatgtca acagcgttcc tagagaagac aggctggaag atagctgtga 16810cttctatttt aaagacaatg ttaaacttat aacccacttt aaaatatcta cattaatata 16870cttgaatgaa aatgtccatt tacacgtatt tgaatggcct tcatatcatc cacacatgaa 16930tctgcacatc tgtaaatcta cacacggtgc ctttatttcc actgtgcagg ttcccactta 16990aaaattaaat tggaaagcag gtttcaagga agtagaaaca aaatacaatt tttttggtaa 17050aaaaaaatta ctgtttatta aagtacaacc atagaggatg gtcttacagc aggcagtatc 17110ctgtttgagg aaagcaagaa tcagagaagg aacatacccc ttacaaatga aaaattccac 17170tcaaaatagg gactatctat cttaatacta aggaaccaac aatcttcctg tttaaaaaac 17230cacatggcac agagattctg aactaaagtg ctgcactcaa atgatgggaa gtccggcccc 17290agtacacagg ggcttgactt tttcaacttc gtttcctttg ttggagtcaa aaagaaccac 17350ttgtggttct aaaaggtgtg aaggtgattt aagggcccag gtcagccact gtttgtttac 17410aaaatcaggt aactaactgc atacactttt tctctttcca tgacatcaag actttgctaa 17470agacatgaag ccacgggtgc cagaagctac tgcgatgccc cgggagttag ccccctggta 17530atagctgtaa acttccaatt tctagccata cgctcagctc atccatgcct cagaagtgca 17590tctggagaga acaggtttct aagcataaaa gatgaaagag cagttggact ttttaaaaat 17650tcagcaaagt ggttccctct cttagggaca gtcaaaacca agtcacttag gtagtaccaa 17710aataaataag gaaaagctta gctttagaaa cagtgcaaca ctggtctgct gttccagtgg 17770taagctatgt cccaggaatc agtttaaaag cacgacagtg gatgctgggt ccatatcaca 17830cacattgctg tgaacaggaa actcctgtga ccacaacatg aggccactgg agacgcatat 17890gagtaagggc actgacggac tcatgatttc ttcttaccag atgctttcct gttctttaag 17950agtttaaaat catcagaaag gaaaaacaaa ctctatattg ttcagcatgc 18000 <210> SEQID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 18 ctttcagaat ctgcctctat 20 <210> SEQ IDNO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 19 agtccatccc atttctggct 20 <210> SEQ IDNO 20 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 20 actgtggtga gtctcccacc 20 <210> SEQ IDNO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 21 agtgtcccag tggcgacctg 20 <210> SEQ IDNO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 22 cacagtccat ggcctgggca 20 <210> SEQ IDNO 23 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 23 ctccgcttcc tcactccgaa 20 <210> SEQ IDNO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 24 tactcgggag gctgaggcag 20 <210> SEQ IDNO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 25 ccgtctttac taagatacaa 20 <210> SEQ IDNO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 26 tcaagacagt aaatcctgca 20 <210> SEQ IDNO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 27 ctttttagat cacaggaaaa 20 <210> SEQ IDNO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 28 gccatttaat tccaagaata 20 <210> SEQ IDNO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 29 ggcccactga gtggacagct 20 <210> SEQ IDNO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 30 gcatctgttg tttaaagcca 20 <210> SEQ IDNO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 31 acggagcagc cgcatggcac 20 <210> SEQ IDNO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 32 ggtttcacca tgttggtcag 20 <210> SEQ IDNO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 33 tctcggctca ctacaacctc 20 <210> SEQ IDNO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 34 agggacgctg agatctgcgc 20 <210> SEQ IDNO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 35 ggtctcaaca ggcagaggca 20 <210> SEQ IDNO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 36 atccctgagg ctggaaccgt 20 <210> SEQ IDNO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 37 caaacaccag taggtttgtg 20 <210> SEQ IDNO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 38 gaagccaaac accagtaggt 20 <210> SEQ IDNO 39 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 39 tgcggaagct gttgtcagaa 20 <210> SEQ IDNO 40 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 40 gggagccagc actggcagct 20 <210> SEQ IDNO 41 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 41 cgggagtggc tgctgcggtt 20 <210> SEQ IDNO 42 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 42 gctggacctg ggtttcctca 20 <210> SEQ IDNO 43 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 43 aagcagcccc ttggcaaagg 20 <210> SEQ IDNO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 44 agggctggat ctggaagtgg 20 <210> SEQ IDNO 45 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 45 agaaggcaga gacattctca 20 <210> SEQ IDNO 46 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 46 gcccttcctg gaccttccca 20 <210> SEQ IDNO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 47 ctcagtctag aggcaaaggc 20 <210> SEQ IDNO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 48 ctgatccgtc tgtgtccagc 20 <210> SEQ IDNO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 49 aagtagctgg gattacaggc 20 <210> SEQ IDNO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 50 ggccctgtac ctagctccca 20 <210> SEQ IDNO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 51 atcataccac tacactccag 20 <210> SEQ IDNO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 52 ttgtatttta agtagagacg 20 <210> SEQ IDNO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 53 acaaggccag cccccactgg 20 <210> SEQ IDNO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 54 ggcagagaca gagcagactc 20 <210> SEQ IDNO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 55 tgcctggcaa tattccggat 20 <210> SEQ IDNO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 56 cccgacctgg gcgaggtgcc 20 <210> SEQ IDNO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 57 gatgctacgg tccatgctgt 20 <210> SEQ IDNO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 58 acctcctccg accggctggt 20 <210> SEQ IDNO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 59 ccagggcagt ggccaggtcc 20 <210> SEQ IDNO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 60 ctagggtagg cctgcagcag 20 <210> SEQ IDNO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 61 tgtctctagg gtaggcctgc 20 <210> SEQ IDNO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 62 cggagcaagg acggcgtgtg 20 <210> SEQ IDNO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 63 aaattcactg ttgtgtgaaa 20 <210> SEQ IDNO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 64 tgcgtaggtt ctggttaata 20 <210> SEQ IDNO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 65 agagcagtgg gatcacaggc 20 <210> SEQ IDNO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 66 tgttggccag ggtggtctgg 20 <210> SEQ IDNO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 67 agctgtccat acagactgct 20 <210> SEQ IDNO 68 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 68 cttctggaac tgtccgttca 20 <210> SEQ IDNO 69 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 69 gttgacatgc cagggctccg 20 <210> SEQ IDNO 70 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 70 atagaagtca cagctatctt 20 <210> SEQ IDNO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 71 tgtagattta cagatgtgca 20 <210> SEQ IDNO 72 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 72 ttaagataga tagtccctat 20 <210> SEQ IDNO 73 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 73 tccttagtat taagatagat 20 <210> SEQ IDNO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 74 tagttcagaa tctctgtgcc 20 <210> SEQ IDNO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 75 ccggacttcc catcatttga 20 <210> SEQ IDNO 76 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 76 aaaagtcaag cccctgtgta 20 <210> SEQ IDNO 77 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 77 aagttgaaaa agtcaagccc 20 <210> SEQ IDNO 78 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 78 gtaaacaaac agtggctgac 20 <210> SEQ IDNO 79 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 79 gtatgcagtt agttacctga 20 <210> SEQ IDNO 80 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 80 tgatgtcatg gaaagagaaa 20 <210> SEQ IDNO 81 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 81 tttagcaaag tcttgatgtc 20 <210> SEQ IDNO 82 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 82 tgtctttagc aaagtcttga 20 <210> SEQ IDNO 83 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 83 aacctgttct ctccagatgc 20 <210> SEQ IDNO 84 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 84 tagaaacctg ttctctccag 20 <210> SEQ IDNO 85 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 85 tgcttagaaa cctgttctct 20 <210> SEQ IDNO 86 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 86 aatttttaaa aagtccaact 20 <210> SEQ IDNO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 87 tgttgcactg tttctaaagc 20 <210> SEQ IDNO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 88 agcttaccac tggaacagca 20 <210> SEQ IDNO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 89 gggacatagc ttaccactgg 20 <210> SEQ IDNO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 90 tttaaactga ttcctgggac 20 <210> SEQ IDNO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 91 gacccagcat ccactgtcgt 20 <210> SEQ IDNO 92 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 92 gaagaaatca tgagtccgtc 20 <210> SEQ IDNO 93 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 93 gattttaaac tcttaaagaa 20 <210> SEQ IDNO 94 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 94 tagagtttgt ttttcctttc 20 <210> SEQ IDNO 95 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 95 aatatagagt ttgtttttcc 20 <210> SEQ IDNO 96 <211> LENGTH: 30310 <212> TYPE: DNA <213> ORGANISM: Mus musculus<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (19791)...(19802)<221> NAME/KEY: CDS <222> LOCATION: (21160)...(21370) <221> NAME/KEY:CDS <222> LOCATION: (24168)...(24307) <221> NAME/KEY: CDS <222>LOCATION: (25696)...(25908) <221> NAME/KEY: CDS <222> LOCATION:(27235)...(27246) <400> SEQUENCE: 96 gctcgctttg ggtcatgatg tttcattataggaatagtaa gccaaactaa gatgatgtct 60 cttcacaaca ttagaaaagt gactaagactggcctctata gactcatacg tttgaataga 120 actatttggg aaggactagg agatatagccttgttggaga aggcgtgtca ctgagggtgg 180 gctttgaggt ttcaaaagcc cagagtctttccttctctat ttcctaactg cagataggga 240 tgcaagctct cagtgattcg ccaccaccatgtctgcctgc ctcttgccac gttccctgcc 300 atgatggtca tggactctaa ctctatgaaaccataagccc caaattaaaa gaaaaaaatt 360 gagagagagt ttttttctgt atagacctgactgttccaga atcactcggt acgacacgac 420 gcgaagctgg ccttgaactc agggatcctcctgcctctgc cttccaagtg ctgggattaa 480 agggatgtgc caccactact caactaaatggtttctttta taattcatcg tggtcaaact 540 gttttgtcat ggtaacagaa aaacaactaagacccagcca tgtctgaggc acacacattt 600 atagatgtac agttaagctt tttctaattctgtaatggag acagactcac acaatagtac 660 cgcctggaat gttggggatg ggttctaatgcattatctta attcagctca caaagtcaca 720 tgggaatcta catgttcaca tgctgagggtccctgtcccc agttggtttt tgattgatca 780 ataaagagcc aatggctagt ggttgggcagggagaaagag gcaggacttt taggatttcc 840 aggcaagaaa ctcaggggag aagatgaaaggactctacca tgagaggggt gtaggacgga 900 ccacaccatt gacagggaag cagaaagatcagacttaaag gcctgccaac atgtaagaat 960 ccagaaaggt gactccaggg gccattgattgggtctgggg tcacagagat aaaataaaga 1020 tttgtcaagt attaactcaa gaataccagaggggagtgtg tgctagccta ggggagtttt 1080 ggaaataccc aacgtttgaa ctagtcaagacatctcaaaa tataaaggtt gcatgtatgt 1140 gtctttcatt cgcaaatcca gagagctctggcgggtggct agaagtgtga tcactttctg 1200 ggaactcaga gtggattaac aattcaccattacaagtgca gtttttggta gggaaggtca 1260 tgtttgtaat ggtgccgagt caccaaagaaagagaaacag ctcttagagt tctatgccag 1320 agggcagagg agcatgcaac ccatccttcagggtttgaca agcagaaggc aggctggtgg 1380 cacagaaaaa aatcatagtt ctggactagtctgggctaca tagtaacctc tgtcttaacc 1440 ttctcccctt gccctaaagc atctatgatctgtattggtg ggagcgagag ctgggtggtg 1500 ctgaggttag aaggctccct agctatgggtatttgttaaa atgtgaactc ctccaagaga 1560 tgttataaag tggaaatgtc tagtctctttggaaagttag ttatgacaaa tgacattttg 1620 ctggggcaca caagtgaaag gatgtcttcctaaagcagac acaggaaaga atgttttccg 1680 gaagcagaca caggtaaaag gatgttttgatatagcaaac atgtaaaagg acccttgaca 1740 aaggagtata aatatgaccc cacagaccacaggagatgag cactgagcct tggtttggtt 1800 tgttctgcct cgctgttctt cgctaactacatacatgcat tggtttacct tatatagtgt 1860 tgttaatcgc aacttgtgga aacaccaccattgagagaaa gagcagtcca ccaaagaact 1920 gcttgtgagg ttcctacagc agcttgctgcttctgcggcc tcgcctcagg ctgcttggtg 1980 agcctagcag tttcttcgac tggactgtccttgccagttt gtgtgtggtg tctgtctgct 2040 tagaagtctg atctgcagct gctgagttctatttggcgtt tgctacgaga ctgaactgcc 2100 cccaaagaac tatggcaccg tccacttcccccatagccta attttctctt ctcccacctc 2160 tgctgggtgg tgggctagag gagacgttgaacctttatta aaagtaggtt gcaaaaaagt 2220 tgagcctaca aggttatata ttcagaacaatttctggaat acgattgggt ctacgtggtc 2280 ctagaaatat tcaggggcaa agaacacgcagcttgtgtgc gccaggttct gctggctggg 2340 tggagagagc gtgccaggta gcacagtgtgccaggcagca cagagccttt gccctctccc 2400 accctagccc atccctattc cttgtgtcacaggaagtatg gagctaggac cagggaggtg 2460 attgttctgt gatctctaat gtttaggtgagaaatgcccc ttcacaccag acctttgtgt 2520 tcacaccagg cccctgggtt cacaccagttacacttattt taatgaagct ctttctgtct 2580 aaaatttcta gctcctccct ttaacacttcctaatttaga gattatttag gctgcacatt 2640 aaaactggaa gtttcactga tagttcagtggtaaggttgg actcatttaa agtgaaaatt 2700 ggattcccag caaccacacg gtggcccacagccatctgta atgggatccg atgccctctt 2760 ctgatgtggc tgaagacagc tacaatgtactcatatacat aaatgaataa ttaaagtgaa 2820 aattggtatg ttccatcttt atgaagttgtgaaatcagtt tccctttttc atttgcattg 2880 attgccaagc acctcggaga gaatcccagttaaaaatatt acgtgttcag gtcatgatca 2940 tgcacgcctt taatcccaga ggcaaaagcaggaggagctc tgtgagttct aggccagcct 3000 ggtttgcata gctagttcca ggccagtcagggctacatag tgagagcctg tctaaaaaaa 3060 aaaacaaaac aaaaacaaaa cttttttctcattattttcc actttgaaat ctagataatt 3120 cagcttgcat gttttaaatt taaaaactctgtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 3180 tgcctgcata tatgtgcacc acatgtgtgcctggtcctca tagaggccag aaggggggtc 3240 agtcccttgg aattagaata acagatgattgtcagccacc atatgggtgc taagtactga 3300 acccagatgg atgctctgta agagtgagaagtgcttttaa ccagtgagcc atctctccaa 3360 ccctgccccc gctgttcatc accaagctcttccactatgt gatttcaagt gtaacttttt 3420 ttttggcggg gggtgggggg gtgggggagtggggggtggg gtggggttgg tttttcgaca 3480 gacagggttt ctctgtatag ccctggctgtcctggaactc actttgtaga ccaggctggc 3540 ctcgaacaga ggcctcccaa gtgccgggattaaaggcgtg cgacaccacg cccggcttca 3600 agtgtaactt ttattgatcg taaaattagagccatcttcc tttaagaaga attggaaaat 3660 ataaagagga aaaagaaacc ctggagatggctcggtttgt aaagtacttc atatgcgtaa 3720 gaactggact ttggatccct agcacccatgtaaaaactag agtgctgtgt gtatctacaa 3780 ttccattgtt attggtgcac ggtggaagcttcctggagct cacctggcag tcagcctagg 3840 gaaatcacgc gtggagctgg gaagctggtccactcccctc accccacacc atctcaaaag 3900 aaaaaaaaaa aggtggaaag gtggagagtgatgaaggaaa acactgacct ctggcttaaa 3960 tacacacata cacacacaaa cacacaccaaccatgtgatt tttttttttt tttttgtctt 4020 ctcagatcca gtttctctgc tcaggaacagcaatttccat ggttctattt acttcctcat 4080 acttccagaa ttcactttct tgtttctctttcacttttgt cactgccacg tgtcctttgg 4140 gggtactggc tggcacttaa gtatatagcattgggacttc tctggacagg ggaactagct 4200 agcagtttga gattatctgc tagcctcctggttctttcca cattcatcct tgctgattca 4260 ttccatgacc gagaaccccg caacccccatccctgccttc cccacaagag tttaaaaatt 4320 ctgcaagcag ctgcgcagga gaaacaatagggacctccca gcatctctga tagggccgat 4380 tctgacaggg tcactagtct tgagtgtgccaaccctgcta tgtaatacat caagacaatg 4440 cggagaggtc gggatcaagt atgacaccccatcctcacga gggcaggtcg cccaggcttt 4500 ggggactctg gggagcgcag gttccgggtgtaccttcctt cctgtccccc gtagcgagcg 4560 ggtaggaccc ttgggtttcc gcaaagtgtggccagtcgga gggcggagca tccggagggc 4620 ggggctatca caggggcggg gctcccgggcgagcacgagg aaaggtaggt ggagtagagc 4680 gccgggccga gtgtggctcc gcaaacctttgccttagccc gttcgccgcc cggtaccggc 4740 gcagcggcgt ctgcgtggtg agtatgcccaccctactggg cgcccccacg gttcccctct 4800 gggaggacgg ggtcggcacg gagctcagtttcgtatgcta tcgatccttc gtgatggcgg 4860 ggctcttgcg ccttgatgga ggcggggtgggggcgccggc cacagggtgc caccgcggag 4920 ctgaggggaa ggcactcact cgaaggcctggggcgtgcgc cactcgcggt ccccctcagc 4980 gctcggtcct ggtccgcttc gggcaggcctcctggtggac ccggggtccc cgcggtcgcg 5040 cgccactcgg caggtgcgcg cagagctggaaaggcgggcc tgaggtctcg ctgcgctccg 5100 ctatggccac ccacaaaaat caacaaggaacggctacagc ccacaaatgg gccctgcaaa 5160 agccctggaa ccccaaccca gggaacacagaccttggaag actgcagcga ggggcacctt 5220 tcctacaccc gtgggcacta ctgtgtgcacagctcacact cacgcctgaa ctgtgaggaa 5280 gtggctgacc cctccgcatc tccagtacccaaaatggttt gaaaatgtgc acagactggt 5340 tgctgatgtt tttaaaaagt ttgttgaatggttggctgaa taaccctata ggattctaga 5400 agaaacccac agccttcagc caccaagtggcctgggccca caaggattca cacattcgtt 5460 cattcattct ttcgtacatt catttacatactcaacaaat aagtgtggac cagggacgga 5520 tcagggtaga actttgtggg tggtgagaggctggaatgaa gagctctgta aaggaccagg 5580 tggtgttgag tatgggactt ctaggctgggcttgaccttc atctgataag ccacatagtt 5640 ctgagtcaag agcatcctga ggacccaggcagggctcccc tactttccca ggctactgcc 5700 tggtgccatg gccaggattg cccttactggaagactacct tgaagccggg tctaggataa 5760 gctagctgtg gaatggagct gggagaaaccacaagaagga tgtggacttt ccacattcca 5820 gctctaccca accaggagac tttgcagccctgccccatcc cctgggactt ggtcccaggc 5880 actaccctgg cagtcagctc tgagtgtttccatggggggg ggggggggag cctgatccag 5940 tgctggggct gagttcagag gctttaatacttgagtgggc tgagctctaa gaaggactcg 6000 gctgggtggt ggtggggaag cagggtggcgattgtgtgtg tcctggcctc tactgcctct 6060 cttgcccaga gagggaatgg cagggaggttggcttattac agctgggtta gcaggcattt 6120 cacccactga cgaaaggtgc tatctcctggctactgcggg gtggagttgg gtacaggctt 6180 tggtgatggc aagtgaagag aagccggctggatgtggcat gctctataaa gagatttaag 6240 tagccccaag gtggccaggt tactggagctctgaaggatg agttgagggt gtacctgaaa 6300 agtgggctgt tagggcagtt actggcgaggctgggggagg ggaagtgatg ctcacagctt 6360 gaggttacct ggttcctctt atttgcaagaaagaatagcc tacggggggg gggggggggg 6420 cacagtgctg ggtgcctggc ctccggaaggaaggcctgat gacacagcct tttagacctt 6480 ccgaagggca ctgcatgctt ttccagctgcccttttgctc tctagtggga agctgagggt 6540 tggggaccca catctaggct gtgttcaagaccaaagagcc attcctcatc agggagacag 6600 tgaatctgat ggttccaagg atgagagttggaaactgccc gtccataaga agcccccact 6660 gtgggtctgt ggtcactgga cattttgtctgtggttgtat ctctggccac catttgctgg 6720 gccgtggctg tggagggcag ctggtgtttctgtttctttc tgggcacgct gcctggctgg 6780 ccagtctcag aggccacatg tatttttcctcatagtctga aggagacaga taaactgaag 6840 cttcaggttg gagggcagtg atgggcaagtgctatacaga gccttctggg tctgataagc 6900 ccacagagag ctttgttttc cttctcaaatttcttttttt aaaaggcaga atgtcgccca 6960 gacttgtctc caactcctgc tcaacaatacctccttgctg ggccgtggtg ggacaccttt 7020 aatccaagaa ctcaggagac agaggccagtggatctctga gttccagcca gggctgtaca 7080 gagaaaccct acaacaaaca aacaaacaaaacaaaacaaa agagtacttc ctgcctcgtc 7140 ctcctaagta ctgggactac agagtgtatcgtttatttta attaactcat gtcgtattac 7200 aataattaga gactagatta ttacttccttcttcagaaag gtacattggg cagagagggg 7260 ctaatttact tacccagggt ctcaaaatcaggtggaaaac tcccagttta actgtaccac 7320 ctgattctca ggctgcgctc tgcttcccaagggaggtcca tctgtggagc ccaatagtcc 7380 tcgggggtaa ggaacagaga ggatgcccacggtgttgttt gcttttttaa cactagggaa 7440 aaccccggcc tagtgtttgt tccatgtgcattctgccact gagtcagaca tgcacagccc 7500 cttcctgtgg actcttcccc ctagcaggtagagggagaca gggcagctag gtgtatgaat 7560 ggggaagctg gaactttagt gccagggacctttatggtgg ggtttccccc acgaaccatc 7620 ctggcagatg tccacagcag atgtgtctccagttcactgt gtcttactct ctgactcttc 7680 tccctcgact ttcgctggtc caaacagggatatttccgac aaaagggtgg tagcatctac 7740 cctgagctaa acaagatgaa aggcaaccatttctagaggt gctgccatct tgaaaattga 7800 gttcttagtt ggctttatgg gcatttatcctcacagacat gttagccttc caaaaacatt 7860 caaacaaaac caagtgaaat caagggaacagaaaacagag gacaagtgtt ttgtgctctc 7920 ttctcttctc ccacccctct ccctctccctctccccctcc ccacctcccc ctctctgtct 7980 ctgttggtgt caagtgactt cctcagtcattctctacatt tccctgtgtg tgacaggact 8040 cttcactcac cgatttagta gactggctggccagtgggct ctagggatac tccagtctct 8100 gcctccccag cactcggatt ctaggctcagagcactacac tagccttccc atggtcctcg 8160 tgatcccagc tcagacccct atgcttatataggcctggag tttacagact gagccatatc 8220 ctagccctgg tttgccttaa gttacccttcttccccagta atgcaaacag acattaggaa 8280 gtacttagga gccaggtgtt tccctactggcccctggatc ggcctaagaa gggcagtgtg 8340 ctttctggca ctatgcctgg aagggtgaggatagctaaac cctggcccag gactgggctg 8400 tgtggaagaa ggcagccaaa tgtagagagagtttgcctat ctgtgtgtcg tgagacacag 8460 gacagatgct tttttgcagt ttcctgcatagtttctctag tctggaggga tctcctggcc 8520 catagtgggt ctactgtcac catgatggccacagccaggg aaggcctgta ctgccttagg 8580 ctactgttcc ctccttcagt gacaaaccttctttgttttt gatttttttg ttttgttttg 8640 ttttgttttt ttggttttcg agacagagtttctctgtata gccctggctg tcctggaact 8700 cactttgtag accaggctgg ccgcgcctagttttgttttt gcttgttttg ttttgtttta 8760 tgaggcaggg tctcacatat acctgaggctggtttttgtc tcactatata cctgaggcta 8820 gccttgaaca cttgattctc ctgcttccagcttcccaagt gccaggatta caggcttcaa 8880 atctttcttc agaggcagta aaagaacagctgaagcctgg gtactcgaga ttccagcttg 8940 tgtgatccag agcccttggc tgtaggcttttacctgagcc agcagtttag ttttcataac 9000 tggtgtatgc atacatgttt ctcctgtagtggtgctgttc ccaataagta cgttacctca 9060 gcccacctta tgtgtcctca gaacagacagctagccttcc aaggacaagt gtgactgatg 9120 ggggaaaagg gaccctggaa ctcaccagagccaccctcct ctagctgagg acatagaaaa 9180 cctttacctg gatttctgtg ggaacttcccaacaggcttt tcctaaccag tcttggaaag 9240 gtgtattgag actgggtgac accatctggaagaggccttg gaacccatag gagcctacca 9300 tgcctcctca gtctggcgtg ttgctatcttatagcataga cctatcttcc cttgagttct 9360 agacaaggca agtttctggc caggacatggtcttgttttt ctttgagcat cttctagaaa 9420 ccagggagac cataccacaa agcccttactggactgacta ctgcatgcgc acctccagga 9480 gcccatctca tcaggcaagg tgactgctgtcctgtctctc tgatggaggc cattgcccct 9540 ttaacaaacg aataaaggtc gctctcccctctagggtgtg gaagacagga aatggctgtt 9600 acccaatgca ggcccactgc cagctctgccctcagagcac ggtgcagaca gtccagtcgt 9660 cctccattgg attctctgct gggctaggcacccccagtcc ctctgtggct aagctaagaa 9720 aaagagagag aaaaaaaaaa aaaaaagagtaaagcattgg gggtggggca ggaagagagc 9780 acaggcgtgc aaacatcgaa gagcggcctctgtgacatct gtctgcgccc ctgttggctc 9840 acccttagga catctgactc cctttctgctagccatcttg tcccacccaa tgcttagata 9900 tttcagaagc ctcggtcctg ggtagggagggaaagcaggt ctctgtatct tataggcctc 9960 agacaaccag gacagccatc ttctgcaggcctagtgaggc cccagggatg ggcagcttca 10020 gtggcatggt gcacacgccc ttttccacaccaccctttgg caagattact ttctgtgcta 10080 atggttaaag gcagaaacct ttgcccactaagcagttgct gcgcccctga gctacgctcg 10140 cgttcttaaa accattgtat tgctggtgtggtgggtcaag tctgtgatgc cagcacttgg 10200 caggccaagg caggaatgag aaggagaacaagtttcaaag caagcctggg cttcatagta 10260 agaacttgtc tccaaagccc aaagaaagggctggagatac aggacagctg gcagaaacca 10320 ggcacagagg ttggcatctg tagacccaacacccggacag tggaggcagg aggatcagaa 10380 gggaagaccg ttcttgcctg aacgtcaagttctaggccag gctgagggcc atgccaggct 10440 ctctactgtc tatgtatgtg ggtgtttgcttacaccactt tcaaacctgg tgcccaagga 10500 ggccagaaga tggggtcgaa tcccctggaactagagttac agacaaatat gagctgctgt 10560 gtaggttctg ggaaatgaac ccaggtcctctggaagagca gcctgtgctt ttaacaactg 10620 ggccattttt ccggcccata ttcatttttattacgtgtag ttgtttattt cattatggga 10680 catcccacag catgcacctg gctatcagacttgcggaagt cagttctttc tttgcccagt 10740 gtgggtccta aggcttcatt cagttcatgttggcaggctt gtgccccctg ctttagatgc 10800 cacgtcatct ccagccactc acatattcttgctacccgtt ccttgtcaga tactttgtag 10860 acgtttcctc cccaggctgg atttgaactcactgtgcaga tccgtctgtc ctgttttagc 10920 ttcctggatg ttgagattac agataggaagcaccatgtct gactcggttt tatcgtctca 10980 ggagtgtctt ttgaatcata aaagttttcaactttgaaga ctacgttagg taattttttt 11040 ttcttttgtt acttgtgcct ctgggctatgtctaagacgt tgcctaatac aagataattg 11100 agacttcctc tcgtgttctc tttttaaattttttatttta taaattatgt gtatcggtgt 11160 tttgactgcg tgtctgtgta ctatgtctgtgtctggtgcc ccaagaggcc cagaaaagga 11220 cattgggtct cctgagcctg gagtttcagttctgagccag tggatcctgg gaatcaaacc 11280 caggtcctct ggaagagtag ccagtactctactgctgaac cagctactct ccagccccca 11340 cccttcttac acttaggtct atctgttttggtttggtttg gtttttaaga atttgttatt 11400 caggggcaag agagatggct cagcagttaagagcactgac tgctcttcca gaggtcctga 11460 gttcaattcc cagcaaccac atggtggctgaaatgggatc tgataccctc ttctagtgtg 11520 tctgaagaca gtgacagtat actaatacatcaaataaata aataaataaa tctttttaaa 11580 aaataaaaag agaatttgtt attcaaagccaggtgcatct ctttgggagg ctagcctagt 11640 ctacatagta agtttgagaa cagtcagggctacatagtga gacctatctc aaaagaaaat 11700 ctgttattca gactggagag atggtgactcagtggttaag agcactggct gctcttcccg 11760 aggacttgtg tgactcctgg catccacatgggagcacgcc accctctgta actccagttc 11820 caggtcatct ggcaccctct tctggcctccacgggcacca ggcacagaga tacatgcagg 11880 caaaacacca tatacatcaa ataaaaataaaatagtttgt tatctttttt tttgaaaggg 11940 aagacaaagt tttactttta aaaaagattacaagcacccc aaataacatg taacgagttg 12000 agtcctcgca tctcgtgatt tgggataggatacactaaca gcagccggaa taagcatacc 12060 atattgactg tcctaaatta tccaggctagagtactgtaa ggctggctgc tacttcatag 12120 gagttgctaa tagctattac tacttttccataaataacgc ccctgacctt taagaaagta 12180 gaagggaaca gcttactccc tttctttcaaagaatttttt ctacttgact aataaaaaag 12240 tcagcactga tatccattac ttgcagaagacacaggaaac aggtgacaaa cactccttaa 12300 agacacacaa gataagaaga tggaacttcaggtacatagc aagtcggtac aaaaagctag 12360 atttgatact cttaaaacgt gaagggtcctacaacggcat agagaaataa tttaatgcct 12420 tccagaacag aactcgagct ctgtggaggtttcctattct ataggggcag atctcatgcc 12480 aacccacaga gcaggcgctt ccacctcctatccctttatg cggtagcttt catggatttc 12540 tggctggatg tcacacacag aggccaagaggtcattcagg actccatccc tgttctgctc 12600 gaagtggttc tggaggacgt tcatcttcccctgggtctcc tcttccacct cactgctgca 12660 gctgccatga gaccccagtg ctgcagcttccttggcctct gcagtactgt tcaatttcag 12720 cctggcggct tctttggcct gcttcggcctccggttcttt cacttgcagg ccttggacac 12780 cttcttggct gcctgcagta gctgctggatgccccgcaac tgactctcgc cattgctgag 12840 gggactttgg gccgagagaa tggcctaaatcaaccaacgg ctcaaacata gtcagaagcc 12900 cctccgtttg atgtcattta atgagcctttctgtgtagct tcaggtcact ccctgaggcc 12960 tggaacaccc tgaatctttt tcagcttttctgctgaattt ggctgtcacc aggacagact 13020 gctgagggag tgtgttagta ctccagaggagcccagttgt cactatgact ggagcagcgc 13080 agtcttgttt gtggcactgt tgggctatgtctgctcactg acagttggga tcagttcctc 13140 ttaggtgact cataactgtt gcggtaaatctcctcccaaa tatgccccgg caatgaaaac 13200 acaacacagt tcatatgaat acatgctgtgcgcctagatt gggcagatct accgctacac 13260 taccatcttc cacatctatg agaccccttagaacttgcgg tttctccagg ccttgtgctt 13320 ctgctccact tttccccttc tttctccttgtctgtgtcct ctccctcttc cattttctct 13380 ttgttctctc cccccacctt ccgctccaccttccctttta tctgcccaaa cttcagctcc 13440 cctttatttt acaaattaag gtgggaagcaggtttacagg aaatcacctg agtgctgact 13500 atgttcttgt tcacaaccac tctcaggagaacggaattaa catcaaatat aattagcccc 13560 agggctatct gcaacacata acaactatgtcagtgtgatc tggctctatc tgcaagagtt 13620 gaccctctgg tgatgccctg actgagcgtgtcctgcgctt gctaatgctg tggtgctgcc 13680 cctggatggt atgtccacgg ccaacatatgtccaaaagga aagcccctgt cagctgttgt 13740 ttttttcaaa tttatgtcta tgtgtgtgagtattttccct ttttgtatat ctgtgtacca 13800 catgggtgcc cagtgcctgt ggaggcagatgccccagtac tggtgttaca ggcagttaat 13860 atgagctggg aattgaaccc aggtcctttggaagagcagc cagtgctctt aacttctgag 13920 tcctctctgc agccttctta gcatccatttttaatctttt gtatgacatc tggcagaggt 13980 aggaattcat gccttttggg gggatagttggctatcccag tgtccttggt taaaactgtc 14040 tgttctttcc ctggcggtgg cccgggtcagtgtctgatga actcgatgct cactgctctc 14100 tgatttcttc aaccaggccc gcaccttcatgacgtcatga cgagagctat gggaaggttt 14160 gaaatcagga agtacaagtc tgtcatccactttgttcctt ttcaagaatg gcgatttttg 14220 aaaatgtcct ccgcgttcat gtatggatttaggaattgtt tgtcactttc tggagtattt 14280 tttataggaa ttgtgtggag tgctgtagtctgatagtgtg ttgtctcttc cagcccctga 14340 caggtgcttg ccttccgttg tttatctcaacaagttttgc agttttcgtt tagtgtctaa 14400 tgctcgtata acattcgctc ctaaatgctttgtgcattaa ttttgttcac ggcactgggg 14460 ttgctctcaa gctctcggta gacgtgtgttctactgtgga gatgcaggcc gggtcttagg 14520 attttctgtc tcttggtagc acaataatcatttcatttta ttttgggtta tgagtagtgt 14580 atagaaaaac aggacagcag gggcttgctctctgctactt tgttttcttc atgaattcct 14640 tgggtgctgt gtgtaaggtc atgtcagatcactgtgttca ggggcttcca gaagattcca 14700 ctgtgcagct aagcttgaaa attgctgaggaagctgggca ccacagcacc tacctgtctt 14760 cctgaggcct gcaaggtagc gccaagagtagacctcgctg gcggcgtgcc tggcaccccc 14820 cgcctgccat ggaacttgtc ttggtctatgattggtacat gatagacaaa gaggctcttt 14880 tttgtcacat caaggattca gctttgtgaccttaacgttt gttcatcttt atgaataggt 14940 gacatagctg ctttctgttg gggggctgggagagcacacc cggttgctgg actgttttct 15000 ctgcgtcctt ggtcgcaagc tcggttgaactgttttgtgt ccaaggagaa gaacagcatc 15060 cgttactgga cctgtgagtt tgggtctctttgtcctgcct ccctctccct gcctgcctat 15120 gtgtgctcgt gtgtgtgtgt gtgtgtgtgtgtgtgtgtgt gtgtgtgtgt gtgtagaggg 15180 aacctcaatt gagaaaatgc ctccatcagatttgcttgta ggtaagcccg cagggtattt 15240 tcttgattgg tgatggtgtg ggaggtctggcttactgtgg acagtgccgc tcctgggcag 15300 gtggccctga gttctgtaag aaagagcctgagcaagacat ggaacaagtc agtaagcggc 15360 ccccctccct ggccatgact ccagctcctgcctccaggtt cctgccttga cttccctcag 15420 ggggaggggg acggggacgg gacctgagagttgttgtgct gagatatgta cttttctccc 15480 caaattgttt ttggtcaagt gttttattacgatagaaagt aaactaaaac acactctccc 15540 cacacacaca ctgactccac cccacacaccgtgaacacag ggccttgagg attccagaca 15600 gccttgtttt gtatttattt tgggacaaggtcttagaaag ttgaacttgt gatcctcctg 15660 cctcagcctt ttgagtagct gggattataatctgtgtcac cgagtttgtt cttgacctaa 15720 gtagttgaga agagcctttg ctcttgtgtaaatgggaaaa ggtgctttag tcacagaggt 15780 ttaggctctg gcttctcact gatgcagcaccaactggagg agacattcat acaaattaaa 15840 catttttagg atttttaaaa agtgtgtttcaatgttacat ttggggtaag aatgaaaata 15900 caggaattat gtcggtgcat tgggtgttttagattgtgtg tgtgtgtgtg tgtgtgtgtg 15960 tgtgtgtgtg tgtgtgcgtg cacagagttttgaactgaag gttttgctca tgctaagcat 16020 gtgtgctatc acccagttcc tctgaaaaagcatctctaat agaaactgcc cattctcggg 16080 cactccgggt agcagagcag cttccgctactgcgtgttga ctttattgtg ctcttggctt 16140 tttagacatt gtgggaaggg gtggacaaagctcactgttt atgaaacagt ctgggtttgt 16200 gtcattaatg gataaccatg cctattctcgtgcatgtgac cctgtgttaa ttggatgtcc 16260 taccacctaa tgcttcttac aacacttgatgtttactgtt tccaaaattg gacctagatt 16320 tagaaaaaac aaaacaaaac aaaacaaaacaaaacaaaac ttgatttgct tatttctatt 16380 ttgcatgctg gggatggaat gctcaggccttactcttgca ggcaggcatt ctaccatcaa 16440 gctgtgttcc cagccctttc aggagcctgacacctaaagc tgagcttggg caatcctgga 16500 aaatctcagg tgtggccatt tgtattgtaaaaagggaaaa ttagggagag atggagggat 16560 ggatactgga aactgaactc atgtcctctggtaggataga cagaacactt aaccactgag 16620 ccttctgcaa ccccctttag agagagagagggagagagag agagagagag agagagagag 16680 agagagagag agagagcgtg catgtgtgtgttacacacag aggccagaac agctgtcctg 16740 gaactcactt tgtagaccag gctggcctcgaactcagaaa tccgcctgca tctgcctccc 16800 gagttctggg attaaaggcg tgcgccaccacggcccagct ttcaagacaa attcttaacc 16860 gccagtccat ctcgccattc tccaaccagtcccttaaaaa tatttttttt tcaggtgttg 16920 agggtctagc cccgggatac aggcatactaggcacggctg aagcactgag ctccacacca 16980 caattgggta ttattaccgt cttaccctctaggttattga tatgctgcag aatacagata 17040 ttaatgcagg cacttgtcca caggcctttgtccagtgcag tgtggttatt atcttacagc 17100 tattggcagt cttgcctgcg tctctaagttcttctgtttc tcatcatctg tgcatatggt 17160 tctttgtcat ttgagttttg tttatttacttatttgtttg tttattttta tggagacaag 17220 gtattgtata gcccagcctg gcttccagctcacagtgttg aagaaggcgg ccgggaactt 17280 ctgcttcctg cgtgctgcag ttacgggtgtgtgccatcgt ctccggcagc ccggggctct 17340 gcatgcatgt gaggcaggca ctctaccaacagggctgcat ctcaagcacc tgggcagttt 17400 tagcacagtt ccttggtttc ccattaagtaatgagttaaa tatttaacat atgtccattt 17460 gaaaagatgg aaaacaactt ctcctggtcactcggcattc atcagccaga agtctgggag 17520 gctttttctt ctctggatct ccacttggcggcgttctctg cctgctctgt agcctttgat 17580 aagtggatgg ctgggtgccc tctccgtaatatttatcaca tttttctcgg ttacttgtat 17640 agataaacct cagcagggca ggggcacaaggacacccagc tctgtgtaac agtactttgt 17700 accttcctcc ctattggtgt gtcccgagtctgcacttcgg gtgggcgggg ttttgtgaag 17760 ttcagagttt tcagctactt cagggcttttggcttctaca gtacaagaga aacttccagg 17820 ttcctgggag agtgagttgg agtctgagtagtgtgaccca cgtgagctgc tgtccattcc 17880 tcttactcag gacacagctc tctgctcagaaatagctctc tcgtcccaag actccacctg 17940 gtggcttctg gaagaagtgg cctctgtgatggtggagatt gacagctctg actgtgattg 18000 acagctctga ccaccatgag gtgcatgcaaagtgctttca cacctgtcta ataattctgg 18060 atgtaatgag aaataccaag caaggtgtttttttttttaa ttagaatttt tattcatcac 18120 tgtgtgtata tgagggaggt gaactcatgcgtatggaggg aagagggacc tggaaccggc 18180 tcctctttga cctttcacat tgttccagggatggaatgca ggccatctgg cttgctgact 18240 ggcacattca ccagctctct tgcttgcatctgatcttagc ttttttgagg gacctctaca 18300 ctattttcca tagtagccat attaatttgcattctcagta acagtatata caatgaatgg 18360 atatactttt ttaaccatgc aacaaaacctttattaacat tttaaacaga tgttccgcta 18420 ttactgaaac tttgtggggg ttggggcgggggcaggtttc aagacagggt ttttctctga 18480 atagtcctgg ctgccctgaa acttggtttgtagaccaggc tagccgaaaa ctcagggatc 18540 cacctccttc tgcctccagg tgctggaattaaagttctat accaccaagc ctggctgtac 18600 tgaaacttat aatttctaaa ttcaaatgcacaaatggttt tagtgtagag taataccatt 18660 agtgcctacg ggaaatttag gctgaagaacggagaccatg tgtgggcttg agtcttttct 18720 ggatcaaaaa gagtatggtc atctttcagctgcttgcctg taacgatgag cgtctgctgg 18780 gtggggtggg aggtgccctc ctaatcctgggtcttaccct tcacattctc tgtggtatca 18840 gtgggctcta cctcagggtc tgggtcttcacaaagattca catctttttt gggggagggg 18900 gtgcgttgag acagcgtttc tctgtgtagtcctggctgtc ctggaactca ctttgtagac 18960 caggctggcc ttgaactcag aaatctgcctgcctttgtct cctgagtgct gggattaaag 19020 gcgtgtgcca tcatgcccgg caagactcacatcttaacct gttaatgaag ggattaaagt 19080 gcaaagttca aagcacatca gggcacctagttataagagc ctctgcactg gacaaagctg 19140 ctcgtctgga catcctcaat gaagttcttcaatgactttg gtccagtcag ctatggtaga 19200 tcagaagact tgcatggcgg gcacgttttaccagccaagc tgccttgccg gctcctccag 19260 atgacatctt cttcccatta agttggaatacatactgtgt gctttgcctc atcgtgtgga 19320 aagaggaagt ggttggtggt ttgggggcactgtggtcctg tagtgtagat gccctgcagt 19380 cttgcaggag tgtgtgacta gctgggaaacccactaacca gtgtgaggat tagcagcagc 19440 agttcttgtg ggaagcgccg gttggcctgatcagacttac tgaacatggg aagaaagctg 19500 agctctggag aactggcctg gggatgcccaggtcagtgcc agcggaggct tcaaggagga 19560 agactgcaga cctgactcac tgggtctgtgtggagagcaa acaaatgagc caaagccagc 19620 ggtgtggctg ggtgtgcctc agctgcaggtgtgacagtgt cctgtatccc gcggggcccc 19680 gcagaggcat tgctttaggg aacagccacccatggcttgt atatgtcctt tttcaggtga 19740 ttccctggac tctgtgagct ggcagtgcttggagctacac agcttgtgcc atg gac 19796 Met Asp 1 tct gag gttagattctggtatctttt cattttgttc atcctgggtg tccccgttaa 19852 Ser Glu gcaacctgacccctcagttg tcaggtctgg caaggtgtac ctcagataat ccaacagagt 19912 tcatctccactggcacctga tagggactta gtacagaatg gggaaggggg acgtccttcc 19972 agaaggacggaacggcgtga ctgtcagctt ggtagacata gcaagggcgg cacaaaggcg 20032 ggacagaaaagatctggaag gttccctttt gccccagtca gggggctgag ctgggctcgg 20092 gcaatagtgctttctagcct cccagtatct cctgctgtcc tgcagggcct cttgagagtg 20152 ggcccctcctggacaacggt agacttgctg ctgtcccctt cttctacctt ggagcaggaa 20212 agctgaggcacagaagaaag tgaaatgctg acattttctc ttacatcttg gcatttgaca 20272 tccttgccccacatcagaac ttgtatctta ttgtagatgt ttctgacttt atgacaactg 20332 ttatgcacacagttgaggga cattaagtga agcaggtttt gctactacgt ttttttgtac 20392 tacagggactcatggaacag gcgttgctga gtgctcctcc ttttttttgt tttttgtttt 20452 tttcgagacaggatttctct gtatagccct ggctgtcctg gaactcactg tgtagaccag 20512 gctggcttcgaactcagaaa tccgcctgcc tctgcctctg cctcccgagt gctgggatta 20572 aaggcgtgcgccaccacgcc tggcgctaag tgctccttca tagtgctcct acccagggct 20632 gcttttgtacacaccataga actggcagag aggccggtga gcaagaccct ccctgctgcc 20692 tctgatagtgcacatgtccc cctgaaaggc acaggcagag tcggacctgg gtccctgctt 20752 cctagagtttatcaggcatc ctgtgtctgc tcatgaggga gtgaggggaa agaggaaccg 20812 cttgctgctaggagcacagc ccgtacagtc aggctcagcc ctgaacggaa acatggatgg 20872 aactgaagtagtgacatttg cctgccaccc cagtgtccct gagaccttcc ctcgaagcag 20932 cttccccagtgggtgtcttc aggaggggat ctgtagaagg tggctcgatg gccccttggt 20992 gtcttctgtttggcaagcac accacagcct gtttctctgc ccctgggcct ctcactaggg 21052 catttagatcctccgagtta ttgattgtca caggccattg tgactcgggt ccaactgtgc 21112 tctgacccaggctcccgtga gccttcctga ctccccttcc accttag gtc agc aac 21168 Val Ser Asn 5ggt tcc ggc ctg ggg gcc aag cac atc aca gac ctg ctg gtg ttc ggc 21216Gly Ser Gly Leu Gly Ala Lys His Ile Thr Asp Leu Leu Val Phe Gly 10 15 20ttt ctc caa agc tct ggc tgt act cgc caa gag ctg gag gtg ctg ggt 21264Phe Leu Gln Ser Ser Gly Cys Thr Arg Gln Glu Leu Glu Val Leu Gly 25 30 35cgg gaa ctg cct gtg caa gct tac tgg gag gca gac ctc gaa gac gag 21312Arg Glu Leu Pro Val Gln Ala Tyr Trp Glu Ala Asp Leu Glu Asp Glu 40 45 5055 ctg cag aca gac ggc agc cag gcc agc cgc tcc ttc aac caa gga aga 21360Leu Gln Thr Asp Gly Ser Gln Ala Ser Arg Ser Phe Asn Gln Gly Arg 60 65 70ata gag cca g gtaggtcctg gccttgtcca cctcatccca aatgtagcct 21410 Ile GluPro ttactgaccc ccaaaagcta caagggcttt tggagctcag tctctaacct tacattgtca21470 ggctggtgtg tgtgtgcatg tcatgtgact cctgccttgt gatctgcatg tgactgcccc21530 cagtaatgtc cagttcatat gacatcgcct gtatcaggac aactaattag aaagttcttc21590 cttctgatga gtcctgagtt ctcttcaggt ctggacctga ggatcctctc tggaccaata21650 tttaaaacat ggtttttaaa acatatgtcc caaacagtta tagtacagcc aaagtatgga21710 aattgattgt ctagtttagg cttcattgct gtgaaaagac accatgacca aggcaactgt21770 tttttgaggg ggagggggct tcgagacagg gtgtctctgt gtagccctgg ctatcctgga21830 actcactctg tagaccaggc tggcctcgaa ctcagagatc cgcctgcctc tgcctcccat21890 gtgctggcta ggttttttat ttttttattt ttttttattt cttagttctt tcctgcaact21950 atcaagtcat tcagaaaaga ggagtcaaga gaggggatga ggtacatttg aaataaaaaa22010 ctataatgat gattggtcct gcttctgcct ccctagtgct gggattaaag gtgtgcgcca22070 ccacgcccag cccaaggcaa ttcttataaa ggacaaattt ggttgaggct ggcttacaag22130 ttcagaaggt cagtccatta acatcatggc aggaagcatg gcagcgtcca ggtaggatgg22190 tgctggagga agagctgaga gctctgcatc ttgatccagc tgtcatcttc cgggctgcta22250 ggaggagggt ctgaaagccc actcccacac ttcttccaac aaggacacac ttcctatcag22310 tgccactatc tgggccaagc atgttcaagc caccatgctg gtcaagatgt tataacccag22370 aagtgccatc agcttcagct tgtggagttt tggaaagtag caaggcagag tccttcgtcc22430 tgccattcag atctgggagg tctgggacat tgctagtctg gtcatggctg ccaggtaagc22490 atccttcaat agccacacag cacctcattt gtgtaggcta gctgaactct caatccagtg22550 aaaactcctg ccgttagagt cattttgcct cctaaatgaa actttaacat atgtgacttg22610 ctattaccta aagagatgac cgagtattga agtatcctga ccctcatttc cagataagga22670 aactgaggca cagcagagaa atggctgacc tcagatcaaa ctgcccatgc agcaggagca22730 aggctcaacc aagctgctcc ttcatcagtg cagtcacctc ctgctaagcc tgtgtcactc22790 ggctgctcct agccttcacc tgtcccctgt cccctgtccc catgctgtgt ttacagcaac22850 tgaggagacc tccctaaagg ctgaggtgca gcgagtgctc agagcgctgt gggcagcatg22910 caggtgggca tcactgagtt cttcagagtg tacaggcctg gctcgggctc tgctcctcca22970 gcaggttctg gagctgcatg atttttttta aaatgcttgt ctgtctgtct gtctgtctgt23030 ctgtctgagt atggggtatg cacatgccct agcatatgta tggagtcaga gctggctgtt23090 ttccttccac catgtgtgtc ctgggatcaa actcaggtca ggatacttca ggactctaag23150 cactgctgcc tccgatcttg gacacagagg cttcactgcc ctctagtggt tgcaagggag23210 accagcagct agtttggctt ccctaccccc ctctggctag tttatttctt ttgagacagg23270 gccttaccct gcctagcctg aaatttgttg tgttgaccag gctaatcatg aactcccaga23330 actctgcctg cttctgccaa atgtggttca tttttcaaat gccctgaagt ggtatcttga23390 gtaggctggg atgtgacagg tattctctac aagctgggtt ttaccatagc cttgtctccg23450 aagcccacca gtgagccagc cagccaggcc aaaactgaag agaagcgcca ggcagtccag23510 gaaaggctca ggaagttcag ggcagcggga ggaggctctg gctgtgcgca ggtgtctgtc23570 actctgtgcc atacccgctt ctttctgcat cagtccatgc cagacttcaa agcctggctt23630 aagtcacgag actggggatg acgaggcttt gcagacgatc gatcggctgc agattgggag23690 cagggcaaag tagtggcttc agcaagccag tgagcagctg agtctgccta gaacactcgg23750 ctagtagtgg atttaaatca cagggaaccg gaagccatgc agttactgtc acctaagcag23810 aagcagtgag caccagagag gccttgagga gagcagtgtg gtgaccatgt gacaggcatg23870 gactgaggga gggcctggag taccgctgaa tgctgaagca gttgcccact gcattaaagc23930 agcagtgaca caggcaggac acaggacagg agcaccccca accccccagc ccccgcagca23990 gcaagcatat aatctgggac aggcctgctt ctccagccag gttctgctac ccaggccttc24050 cctgcacccg gggaggggcg gcactcatgg tcctcactag ggcaggtgcg gaggtaggaa24110 gtggcctgaa gctgttgaca gaaccattgc tgagtcttgt atttgttgcc taaacag24167 at tct gaa agt cag gaa gaa atc atc cac aac att gcc aga cat ctc24214 Asp Ser Glu Ser Gln Glu Glu Ile Ile His Asn Ile Ala Arg His Leu 7580 85 90 gcc caa ata ggc gat gag atg gac cac aac atc cag ccc aca ctg gtg24262 Ala Gln Ile Gly Asp Glu Met Asp His Asn Ile Gln Pro Thr Leu Val 95100 105 aga cag cta gcc gca cag ttc atg aat ggc agc ctg tcg gag gaa24307 Arg Gln Leu Ala Ala Gln Phe Met Asn Gly Ser Leu Ser Glu Glu 110115 120 gtaagtatga ctctggtctg ggagcccctc ttatgggaca tttcggaagtgtgggacatt 24367 tttccttgtc gaaccagtct ttcccaggaa gtaaaccctg tccttgactgcccgtcagca 24427 tggtctctcc aaagaattta gtcagagtac agagcttagg agtcaggcctccaggaagat 24487 ccctgaagta cctgatctgt acagatactc agtcttctct tgtggcgaactccatgtcgt 24547 tcccccaggg tgagcatctg ctcggctgtg tggttagaat cagcacatggaaaccgatac 24607 aagtccacct cttgctgggt atacggtgaa ggacccaaag ctcgttcctcagcaccgggt 24667 ccttcctaaa gcagaggtgg aggggtggtg gggagagggg agagagagaaaccaaacccc 24727 ggggctgtga agtacctgcc caaggaggaa gattctgttc ttaggacttccagcagctga 24787 aatcgtggct gccctcacca tctagattca ttgtgcctac atacagcctgtctttgctgg 24847 cactctctct acctgccact ctccagtggc tgtcaaagac acacacatttgtcaacagcc 24907 ttgggctcct cctatggggt agattcttta atgtgagcca cagaacctgaagctcacttt 24967 ccaccccacc ttgttttttt gttttttgtt tttttgtttt ttttttgaggcagggtttct 25027 ctgtatagcc ctggctgtcc tggaactcac tttgtagacc aggctggccttgaactcaga 25087 aatccacctg cctctgcctc ccgagtgctg ggattaaagg cctgcactcccctccccatt 25147 ttttaaagag ttaacgttac ctgtttctgc gtgcacctca tgtgtgagtacatgagcatg 25207 cttgcaggta catgcattgc catcagatcc cctggagctg aagtttcaggccattgtgag 25267 ctgttgccta taggtgctgg gaactgaacg ggctcctctg gcagagcagtacatgctctt 25327 caggtccagg ggtccagtat cttcctttcc tgcctgaagg gaagataacatgtagcccct 25387 aaagctaagc tcacagtaac atgagcctaa gatgtgctcg tgtccagccaattctgtaag 25447 catctgagtg cagggaagag ctcagacgcc catatgtcag tagtgtgtacaggctactca 25507 ctaaccatgc actggtgagt ctccacgtcc ctctctggtc tgtggagagtgaatcctcta 25567 tcatttcctc cacccaacgt tcttagctat ttaaccacca ctcccctctgaaaggctgct 25627 tcctcctttg gcctgatttg gtctctctga aggaagagca tcagtaaactgtcttcttta 25687 atgtacag gac aaa agg aac tgc ctg gcc aaa gcc ctt gatgag gtg aag 25737 Asp Lys Arg Asn Cys Leu Ala Lys Ala Leu Asp Glu ValLys 125 130 135 aca gcc ttc ccc aga gac atg gag aac gac aag gcc atg ctgata atg 25785 Thr Ala Phe Pro Arg Asp Met Glu Asn Asp Lys Ala Met LeuIle Met 140 145 150 aca atg ctg ttg gcc aaa aaa gtg gcc agt cac gca ccatct ttg ctc 25833 Thr Met Leu Leu Ala Lys Lys Val Ala Ser His Ala ProSer Leu Leu 155 160 165 cgt gat gtc ttc cac acg act gtc aac ttt att aaccag aac cta ttc 25881 Arg Asp Val Phe His Thr Thr Val Asn Phe Ile AsnGln Asn Leu Phe 170 175 180 tcc tat gtg agg aac ttg gtt aga aacgtaagagcca gcagtgacac 25928 Ser Tyr Val Arg Asn Leu Val Arg Asn 185 190cagcccctgc ctgcttgcct accctattct aatgcagcag agcctctgct gaagcccctc 25988tggcccgctc tcccttttga ccacccgcag actgagagag gcaaggctgt ttcacaccac 26048tgatgggaat cgagcaagct ggggggacgt ggagtgttta ggaagatgac taagggctca 26108gccccctaag tgtgtgtggt gtgcacatgg aagccagagg tcattattgg gtgccttttt 26168atctcgctct acctatcttt gtgaggtagg gttggttctc cgtgaagtca gaacttgccg 26228gttaggctaa actagcaaac cctgggcttc cactgcctgc cttcccttcc ctcactgggg 26288taccagttgt ttaatgtgta ttgatgctct acctgaatgt gtgcctgtgg accatgtgtg 26348cctgatgcct ggatagccag gagggtgctg catcatctgg gattgagttg caagtggttg 26408tgagctgcca tatgggtgcc aggaatctga actcggttct tcaggcctct gtagctctta 26468ctgagccatc tgcacagccc caggtattat gagtaatcag aaagtgacta cacttatttg 26528tgtgcgcatg ttgctgtggg agcatgtgtg ctacagcata gtcggtcagg acgactctga 26588ggtcccaggg attgcactca gctcatcagg cttggcactg taagccatgg cccatgactt 26648agattctttc gaagggcgct tcccgaggat ggagagagaa actgatagga gtaataaatg 26708agttaagtga gaatcgctgt caagctctcc agtaagcctg aggacgggcc cattgctagg 26768gtagccctga gtttctattg cgcatgctca ggaagtggtt acacggagct aagcccaagg 26828tcagtctact gagactgctg gaaaatgacc acgtgttctt agagtcttgt gctctggtta 26888cacaaaccca agtgggagct ggatggagat acctaacctg cactaggatt ttacaatgtt 26948tgggatttta gaacctgtca gaaacattat ccgagattct tttgggggga gggggttttt 27008gtttattctg ggtgaaggca gagtccacat tcccagatgg caatggaatg caaggcaatc 27068ctcctgcctc agcatctgca ggcatgcacc cccacacctg ggtgggtgga gcagaggaca 27128ggtctctgtg tgccaggcag gcactgttga ctgagcagca gcccagtgct tgttttctaa 27188cgcaccgtat cctccaatga gacttactct gctgcctctt tcttag gag atg gac 27243 GluMet Asp 195 tga ggagcccgca caagcccgat ggtgacactg cctccagagg aaccgcgacc27296 atggaaagac cttggcctga agacaggtcc cagagaacag ctgtctccct atttccaggt27356 ggtgggaacc ccaagctggt gattcactgg acatctctgc gttcagcttg agtgtatctg27416 aagagtttac gccggctcct gcatccacac catgtacctt tgtcctatca gctgtatggg27476 ttcccacttg ggaatgaaac ttaacagcag gctgtaaggc agaaaagcat ctttgtaatg27536 ccaagtgact gttcctgaga gccagctctg ggctgtcttc accatgtagg tgggcttctg27596 tctaaggaga acagcattag gagaggtgca tcggcccatg agcgtgaagt ccacccagcc27656 tagtggacac tgaagtgctc acaaggcctc cacctgcctt tgtaaaagcc gaatggctga27716 tctcaaacca tgggaagccc gaccgcccca cccctcctca ccccagcgtt tagctgtttc27776 aggggtcagc tattatctca agatttctat ccaagtggaa acaaactgaa tcatgcacac27836 gacttatctg tgtggtgtca gttacactca ggctcttgct acggaatgca aagaacaact27896 cacataccag tgtcaaacag aatgcacaga agagacctaa aacagcagca ggtcactcgg27956 ttcacaaaag gtgactccca gtcaggtctg acactgtctt ggttgtagag cacagctgcc28016 atcctctttc cctgggtaac atcacagaag attccatatc aaaagcaaat gttccctccg28076 cttctgtatt tcagagacaa ggcctcactg tatcctcaag cgttgttacg tcttgtgctg28136 aactttgctt aaagctggga tcgtcagcac gagccgccac agcctgcaag tattctagtt28196 ctgaactcat cccagccatg gtggctgtga tggcttgggt gtatcatacc tgtaaattag28256 tggatttttc tttaggaaca tgacctttgg gtgagtataa ttgagaaatt attttaattc28316 agaaagtact tttcattctg ttctaaaaat atgtgaattg tcttaagtgg tagaaatttg28376 tttcttcaaa ataaaaggct cttctctaga tgtttgggag agctgtatct ccaaatgacc28436 tagtacatca gaaggtcaga ccatcccagc agaaacacac agctgtttgg gtcacagttc28496 tgagggctgt ctttattcca gcgacttcac tagctctgct gactggggac tgaggtgtgg28556 ttttgtatcc caggaccatg ttttcaacac tgaaaggcaa accaagagtg catgcacttt28616 tagaatatga aacgtgacct gaaataatcc cccaagtaaa tagtggacaa aaagatgagt28676 caccagttat cataaaatct cgttttattg tcacctccag ggtgcttccc cccatgatgt28736 tgcttctaaa tgaaagcaca gtttgtagac ttgaattgtc acttgccgat aaagaataga28796 ttgggcacaa agtagacaac agtatgggaa aggggccgga acaattggaa caattcgcag28856 taatagagtg agcagatcag acagcagcag tcagctgttg gcgcacactg caaatgaacg28916 ctgcctgggt taaatgctta tgctagttta gttttttttt ttttaagata ggatctcaag28976 tgtccagggc tagcctttag ctctgagcct agtatggcct tgaacattgt cttcctgcct29036 tcacccgagt actgggatta caggtacgta ttccatgccc aggatggaac ccaggatttc29096 atgcaccccg ggcagacatt gatagctaca tctacctgac tctgctatgt taaggataac29156 cattccagta cctgggggac aagataccag aaccactaac aaactgagtt taatcaagga29216 gttaggagaa agaggcactt ttagtctcaa ggaagaaaat catgggttgt cagagcaggg29276 gaaatacagg tccaggagaa aaaggctggc caacagatgg cccatggatg taggaccaca29336 cagactgttt taggcctcac taagggaggt gtgtagctca ccttcctggg ggaaggcatc29396 cacaaacctg tcatctcaca atgacaaaac gtggcactgg caagaaaact ccatggatca29456 aggtgccttc catcaagcat tgggacccac atatcggaag tagagaacaa accaacttca29516 caagttgtcc tctgactccc acatgcacac tgtggcatgc agccacacac acataaataa29576 atgaacagct tttcgtatca aaatgtttgc cgaaagctat ccagtaacca gcttattatt29636 ccgtgccgca aagggcagca ccagagtgac gtgctgacgg aggcccctga gctgactgct29696 aatttgggcc tcggcctcaa aggtgtccct gagacggttc tgacctgaga cactgacaac29756 atcggagggg atgggggcgt gtgtaaacat gagcatggga aggaccctcg ctgcacacag29816 ggacatggca agccaagttg ggttttcgag gagggctgtg tgaagatgac taggagagct29876 tccagctctc gaatagcttt ttacagggta gataactaag accacagact cgggtctgat29936 gggcacagca ctgttctgtg gcagagtttt cactaggaag cactctcgtc agatgagtgg29996 gatggaaggc tacctcgtta atcctgagcc tgagggccag gaatccaaac agtatctcta30056 ggtgtccact catccttccg tgtgcctacc ctagaccgat ggccattgca gggaggaagg30116 accggaggga tcaaaactgc aacaacaaaa acccgacaaa aatgtcaagt ggctggccgc30176 cttcatatcg ctgcttggtg atgagagctg tgtcagatgg cctgaccttg tttacagcaa30236 gaagacaaca cattcaccaa caacactaca gaccacaggg tcacccagtg cctaaagggg30296 cagtggtgca atac 30310 <210> SEQ ID NO 97 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 97 cgttgctgacctcagagtcc 20 <210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Antisense Oligonucleotide <400> SEQUENCE: 98 ctttcagaat ctggctctat 20<210> SEQ ID NO 99 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 99 ggcccggcgc tctactccac 20 <210> SEQ IDNO 100 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 100 gctaaggcaa aggtttgcgg 20 <210> SEQID NO 101 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 101 cgggtccacc aggaggcctg 20 <210> SEQID NO 102 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 102 gccatggcac caggcagtag 20 <210> SEQID NO 103 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 103 gccaggcagc gtgcccagaa 20 <210> SEQID NO 104 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 104 cttccccatt catacaccta 20 <210> SEQID NO 105 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 105 cacttgacac caacagagac 20 <210> SEQID NO 106 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 106 gaagcctgta atcctggcac 20 <210> SEQID NO 107 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 107 gaccatgtcc tggccagaaa 20 <210> SEQID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 108 gtcagtccag taagggcttt 20 <210> SEQID NO 109 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 109 ttagcttagc cacagaggga 20 <210> SEQID NO 110 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 110 cgcctgtgct ctcttcctgc 20 <210> SEQID NO 111 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 111 cccatcttct ggcctccttg 20 <210> SEQID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 112 ctgaaactcc aggctcagga 20 <210> SEQID NO 113 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 113 ctcatggcag ctgcagcagt 20 <210> SEQID NO 114 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 114 cttgaaaagg aacaaagtgg 20 <210> SEQID NO 115 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 115 tctatacact actcataacc 20 <210> SEQID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 116 ccatcacaga ggccacttct 20 <210> SEQID NO 117 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 117 tccatccctg gaacaatgtg 20 <210> SEQID NO 118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 118 cagagctcag ctttcttccc 20 <210> SEQID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 119 agctcacaga gtccagggaa 20 <210> SEQID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 120 caagcactgc cagctcacag 20 <210> SEQID NO 121 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 121 tcagagtcca tggcacaagc 20 <210> SEQID NO 122 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 122 ttgccaaaca gaagacacca 20 <210> SEQID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 123 gcagagaaac aggctgtggt 20 <210> SEQID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 124 gtctgtgatg tgcttggccc 20 <210> SEQID NO 125 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 125 tggagaaagc cgaacaccag 20 <210> SEQID NO 126 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 126 acaggcagtt cccgacccag 20 <210> SEQID NO 127 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 127 ggtctgcctc ccagtaagct 20 <210> SEQID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 128 cgtctgtctg cagctcgtct 20 <210> SEQID NO 129 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 129 cttttctgaa tgacttgata 20 <210> SEQID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 130 cactgatagg aagtgtgtcc 20 <210> SEQID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 131 ctcagttgct gtaaacacag 20 <210> SEQID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 132 ccacagcgct ctgagcactc 20 <210> SEQID NO 133 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 133 gtcctgaagt atcctgacct 20 <210> SEQID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 134 gaaataaact agccagaggg 20 <210> SEQID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 135 tttcttcctg actttcagaa 20 <210> SEQID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 136 ttgggcgaga tgtctggcaa 20 <210> SEQID NO 137 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 137 cgcctatttg ggcgagatgt 20 <210> SEQID NO 138 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 138 gaactgtgcg gctagctgtc 20 <210> SEQID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 139 cgccacaaga gaagactgag 20 <210> SEQID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 140 aatgtgtgtg tctttgacag 20 <210> SEQID NO 141 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 141 ctacatgtta tcttcccttc 20 <210> SEQID NO 142 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 142 agggctttgg ccaggcagtt 20 <210> SEQID NO 143 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 143 acagcattgt cattatcagc 20 <210> SEQID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 144 gagcaaagat ggtgcgtgac 20 <210> SEQID NO 145 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 145 tgtggaagac atcacggagc 20 <210> SEQID NO 146 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 146 gacagtcgtg tggaagacat 20 <210> SEQID NO 147 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 147 aggttctggt taataaagtt 20 <210> SEQID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 148 gtcattttcc agcagtctca 20 <210> SEQID NO 149 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 149 gcgggctcct cagtccatct 20 <210> SEQID NO 150 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 150 gttctctggg acctgtcttc 20 <210> SEQID NO 151 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 151 tcattcccaa gtgggaaccc 20 <210> SEQID NO 152 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 152 cagaagccca cctacatggt 20 <210> SEQID NO 153 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 153 atgcacctct cctaatgctg 20 <210> SEQID NO 154 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 154 gccgatgcac ctctcctaat 20 <210> SEQID NO 155 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 155 gagcacttca gtgtccacta 20 <210> SEQID NO 156 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 156 agatcagcca ttcggctttt 20 <210> SEQID NO 157 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 157 cccatggttt gagatcagcc 20 <210> SEQID NO 158 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 158 gatagaaatc ttgagataat 20 <210> SEQID NO 159 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 159 caccacacag ataagtcgtg 20 <210> SEQID NO 160 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 160 gtaactgaca ccacacagat 20 <210> SEQID NO 161 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 161 agcctgagtg taactgacac 20 <210> SEQID NO 162 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 162 gtagcaagag cctgagtgta 20 <210> SEQID NO 163 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 163 ttgcattccg tagcaagagc 20 <210> SEQID NO 164 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 164 agtgacctgc tgctgtttta 20 <210> SEQID NO 165 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 165 cttttgatat ggaatcttct 20 <210> SEQID NO 166 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 166 aatacagaag cggagggaac 20 <210> SEQID NO 167 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 167 gaggccttgt ctctgaaata 20 <210> SEQID NO 168 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 168 cgtaacaacg cttgaggata 20 <210> SEQID NO 169 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 169 gctgacgatc ccagctttaa 20 <210> SEQID NO 170 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 170 cttgcaggct gtggcggctc 20 <210> SEQID NO 171 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 171 atacttgcag gctgtggcgg 20 <210> SEQID NO 172 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 172 ctgggatgag ttcagaacta 20 <210> SEQID NO 173 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 173 cacatatttt tagaacagaa 20 <210> SEQID NO 174 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 174 gagcctttta ttttgaagaa 20 <210> SEQID NO 175 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 175 ctacgctttc cacgcacagt 20

What is claimed is:
 1. A compound 8 to 50 nucleobases in length targetedto a nucleic acid molecule encoding BH3 Interacting domain Deathagonist, wherein said compound specifically hybridizes with and inhibitsthe expression of BH3 Interacting domain Death agonist.
 2. The compoundof claim 1 which is an antisense oligonucleotide.
 3. The compound ofclaim 2 wherein the antisense oligonucleotide has a sequence comprisingSEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88,89, 90, 92, 94, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 123,124, 125, 126, 128, 129, 130, 131, 132, 133, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173 or
 174. 4. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 5. The compound of claim 4 wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 6. The compoundof claim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 7. The compound of claim 6 wherein the modifiedsugar moiety is a 2′-O-methoxyethyl sugar moiety.
 8. The compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified nucleobase.
 9. The compound of claim 8 wherein the modifiednucleobase is a 5-methylcytosine.
 10. The compound of claim 2 whereinthe antisense oligonucleotide is a chimeric oligonucleotide.
 11. Acompound 8 to 50 nucleobases in length which specifically hybridizeswith at least an 8-nucleobase portion of an active site on a nucleicacid molecule encoding BH3 Interacting domain Death agonist.
 12. Acomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 13. The composition of claim 12 furthercomprising a colloidal dispersion system.
 14. The composition of claim12 wherein the compound is an antisense oligonucleotide.
 15. A method ofinhibiting the expression of BH3 Interacting domain Death agonist incells or tissues comprising contacting said cells or tissues with thecompound of claim 1 so that expression of BH3 Interacting domain Deathagonist is inhibited.
 16. A method of treating an animal having adisease or condition associated with BH3 Interacting domain Deathagonist comprising administering to said animal a therapeutically orprophylactically effective amount of the compound of claim 1 so thatexpression of BH3 Interacting domain Death agonist is inhibited.
 17. Themethod of claim 16 wherein the disease or condition is a haematopoeticdisorder.
 18. The method of claim 16 wherein the disease or condition isa hyperproliferative disorder.
 19. The method of claim 16 wherein thedisease or condition is a developmental disorder.
 20. The method ofclaim 16 wherein the disease or condition is an immunological disorder.21. The method of claim 16 wherein the disease or condition is a diseaseor condition of the liver.
 22. The method of claim 21 wherein thedisease or condition of the liver is hepatitis.
 23. The method of claim16 wherein the disease or condition is associated with apoptosis.